Truncated NKG2D Chimeric Receptors And Uses Thereof In Natural Killer Cell Immunotherapy

ABSTRACT

Several embodiments disclosed herein relate to the compositions comprising engineered Natural Killer (NK) cells that express a chimeric receptor, the chimeric receptor imparting to the NK cells an enhanced ability to target specific cells, such as cancerous cells or those affected by an infectious disease. Several embodiments relate to NK cells that target cells expressing natural ligands of NKG2D, where the NK cells comprise transmembrane and/or signaling domains that lead to cytotoxic and/or cytolytic effects when the NK cells bind a target cell. Uses of NK cell compositions to treat diseases are also provided for in several embodiments.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/477,335, filed on Mar. 27, 2017 and U.S. Provisional Application No.62/628,774, filed on Feb. 9, 2018. The entirety of each of theabove-listed applications is incorporated by reference herein.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file being submitted concurrentlyherewith:

-   -   a) File name: 44591144002SequenceListing.txt; created Mar. 27,        2018, 186 KB in size.

BACKGROUND

The emergence and persistence of many diseases is characterized by aninsufficient immune response to aberrant cells, including malignant andvirally infected cells. Immunotherapy is the use and manipulation of thepatient's immune system for treatment of various diseases.

SUMMARY

Immunotherapy presents a new technological advancement in the treatmentof disease, wherein immune cells are engineered to express certaintargeting and/or effector molecules that specifically identify and reactto diseased or damaged cells. This represents a promising advance due,at least in part, to the potential for specifically targeting diseasedor damaged cells, as opposed to more traditional approaches, such aschemotherapy, where all cells are impacted, and the desired outcome isthat sufficient healthy cells survive to allow the patient to live. Oneimmunotherapy approach is the recombinant expression of chimericreceptors in immune cells to achieve the targeted recognition anddestruction of aberrant cells of interest.

To address this need for specifically targeting and destroying,disabling or otherwise rendering inert diseased or infected cells, thereare provided for herein polynucleotides, amino acids, and vectors thatencode chimeric receptors that impart enhanced targeting andcytotoxicity to cells, such as natural killer cells. Also provided forare methods for producing the cells, and methods of using the cells totarget and destroy diseased or damaged cells. In several embodiments,there is provided a polynucleotide encoding a chimeric receptorcomprising an extracellular receptor domain and an effector domaincomprising a transmembrane region and an intracellular signaling domain,wherein the extracellular receptor domain comprises a peptide that bindsnative ligands of Natural Killer Group 2 member D (NKG2D), wherein thepeptide that binds native ligands of NKG2D is a fragment of NKG2D.

In several embodiments, there is provided a polynucleotide encoding achimeric receptor comprising one or both of: (a) an extracellularreceptor domain and (b) an effector domain comprising a transmembraneregion and an intracellular signaling domain. In several embodiments,the extracellular receptor domain comprises a peptide that binds nativeligands of Natural Killer Group 2 member D (NKG2D). In severalembodiments, the peptide that binds native ligands of NKG2D is afragment of NKG2D, for example, a fragment of NKG2D is encoded by apolynucleotide comprising SEQ ID NO. 2. As disclosed, herein, additionalNKG2D fragments are also used, depending on the embodiment. In severalembodiments, the intracellular signaling domain comprises CD3zeta. Inone embodiment, the CD3zeta is encoded by a polynucleotide comprisingSEQ ID NO. 13, though, as disclosed herein, sequences that differ fromCD3zeta, but share similar function may also be used, depending on theembodiment.

In several embodiments, the transmembrane region of the effector domaincomprises a CD8a transmembrane domain. In one embodiment, thetransmembrane region of the effector domain further comprises a CD8ahinge region. In several embodiments, the CD8a hinge region is encodedby a polynucleotide comprising SEQ ID NO: 5. In several embodiments, theintracellular signaling domain further comprises 4-1BB. In oneembodiment, the 4-1BB is encoded by a polynucleotide comprising SEQ IDNO. 12, though, as disclosed herein, sequences that differ from 4-1BB,but share similar function may also be used, depending on theembodiment.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to CD8a, 4-1BB and CD3z. In several embodiments, such achimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO.18. In additional embodiments, the chimeric receptor is encoded by thenucleic acid sequence of SEQ ID NO. 108, though, as disclosed herein,sequences that differ from SEQ ID NO. 108, but share similar functionmay also be used, depending on the embodiment. In several embodiments,the chimeric receptor comprises the amino acid sequence of SEQ ID NO.19.

In several embodiments, any of chimeric receptors disclosed herein canalso be co-expressed with membrane-bound interleukin 15 (mbIL15). Insome embodiments, the mbIL15 is encoded by a polynucleotide comprisingSEQ ID NO. 16. In some embodiments, the mbIL15 comprises an amino acidsequence of SEQ ID NO: 17. Other sequences for mbIL15 may also be used,depending on the embodiment. In some embodiments, the mbIL15 isbicistronically expressed on the same polynucleotide as the chimericreceptor. In other embodiments, the mbIL15 is co-expressed on a separateconstruct. In several embodiments, the intracellular signaling domain isfurther enhanced by coupling its expression with that of membrane-boundinterleukin 15 (mbIL15).

In several embodiments, the effector domain further comprises an OX-40domain. In several embodiments, the OX-40 domain is either in place of,or in addition to mbIL15. In several embodiments, the chimeric receptorcomprises the fragment of NKG2D coupled to a CD8a hinge, a CD8atransmembrane domain, the OX-40 domain, and the CD3zeta. In someembodiments, the polynucleotide construct is configured tobicistronically co-express mbIL15. In some such embodiments, thepolynucleotide construct comprises one or more cleavage sites (e.g.,T2A, P2A, E2A, and/or F2A cleavage site(s)) recognized and cleaved by,for example, a cytosolic protease. In some embodiments, the mbIL15 iscoupled to the chimeric receptor by a cytosolic protease cleavage site.In several embodiments, the chimeric receptor is encoded by the nucleicacid sequence of SEQ ID NO: 90 coupled to the mbIL15 encoded by SEQ IDNO. 16 by a cytosolic protease cleavage site. In several embodiments,the chimeric receptor is encoded by the nucleic acid sequence of SEQ IDNO: 109 coupled to the mbIL15 encoded by SEQ ID NO. 16 by a cytosolicprotease cleavage site. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 91 and is co-expressedwith mbIL15 comprising the amino acid sequence of SEQ ID NO. 17. Asdisclosed herein, sequences that differ from SEQ ID NOs: 90, 91, 109,16, and/or 16, but share similar function may also be used, depending onthe embodiment.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to a IgG4 hinge, a CD8a transmembrane domain, the OX-40domain, and the CD3zeta. In some embodiments, the polynucleotideconstruct is configured to bicistronically co-express mbIL15 with thechimeric receptor. In some such embodiments, the polynucleotideconstruct comprises one or more cleavage sites (e.g., T2A, P2A, E2A,and/or F2A cleavage site(s)) recognized and cleaved by a cytosolicprotease. In some embodiments, the mbIL15 is coupled to the chimericreceptor by a cytosolic protease cleavage site. In several embodiments,the chimeric receptor is encoded by the nucleic acid sequence of SEQ IDNO: 100 coupled to the mbIL15 encoded by SEQ ID NO. 16 by a cytosolicprotease cleavage site. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 101 and is co-expressedwith mbIL15 comprising the amino acid sequence of SEQ ID NO. 17. Asdisclosed herein, sequences that differ from SEQ ID NOs: 100, 101 and/or16, but share similar function may also be used, depending on theembodiment.

In several embodiments, there are provided methods for treating cancer,comprising administering to a subject having a cancer a compositioncomprising a Natural Killer (NK) cell expressing the chimeric receptorencoded by the polynucleotides described above, or elsewhere herein.

In one embodiment, the NK cells are autologous cells isolated from apatient having a cancer or an infectious disease. In additionalembodiments, the NK cells are allogeneic cells isolated from a donor.

Also provided for herein is use of a polynucleotide as described above,or elsewhere herein, in the manufacture of a medicament for enhancing NKcell cytotoxicity in a mammal in need thereof. In several embodiments,there is provided for the use of a polynucleotide as described above, orelsewhere herein, in the manufacture of a medicament for treating orpreventing cancer or an infectious disease in a mammal in need thereof.

According to several embodiments, there is provided a polynucleotideencoding a chimeric receptor, the chimeric receptor comprising anextracellular receptor domain an effector domain comprising atransmembrane region and an intracellular signaling domain. As discussedin more detail herein, the extracellular receptor domain serves torecognize and bind ligands on a target cell. The effector domain servesto transmit signals (upon binding of a target cell by the extracellulardomain) that set in motion a signal cascade that leads to cytotoxicactivity against the target cell. In accordance with severalembodiments, the polynucleotide encodes a chimeric receptor thatprovides unexpectedly increased cytotoxicity as compared tonon-engineered NK cells.

In several embodiments, the extracellular receptor domain comprises apeptide that binds native ligands of Natural Killer Group 2 member D(NKG2D). According to several embodiments, the peptide that binds nativeligands of NKG2D is a functional fragment of NKG2D (e.g., a truncation,fragment or portion of full length NKG2D. As used, herein the terms,“fragment”, “truncation”, and “portion” shall be given their ordinarymeanings and shall also be interchangeable with one another. Forexample, in several embodiments, the fragment of NKG2D is encoded by apolynucleotide comprising a fragment of the sequence of SEQ ID NO: 1. Inseveral embodiments, the fragment of NKG2D comprises the sequence of SEQID NO: 2, while in additional embodiments, the fragment encoding NKG2Dis codon optimized, and comprises, for example, the sequence of SEQ IDNO: 3. In additional embodiments, the fragment encoding NKG2D is codonoptimized, and comprises, for example, the sequence of SEQ ID NO: 68.

In several embodiments, the effector domain comprises one or more ofCD16, NCR1, NCR2, NCR3, 4-1BB, NKp80, CD3zeta and 2B4. In severalembodiments, these effector domains are coupled to CD8 alpha.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D coupled to CD16. As used herein, coupled shall be given itsordinary meaning and shall also refer to direct (e.g., a firstnucleotide followed directly be a second nucleotide) or indirect (e.g.,sequences are in frame with one another but separated by interveningnucleotides) linkage of nucleotide sequences in a manner that allows forexpression of the nucleotide sequences in, for example, an in vitrotranscription/translation system, a host cell (e.g., in vitro and/or invivo). As used herein, “linked” and “coupled” are used interchangeably.In several embodiments, the NKG2D/CD16 chimeric receptor is encoded bythe nucleic acid sequence of SEQ ID NO: 23. In several embodiments, theNKG2D/CD16 chimeric receptor comprises the amino acid sequence of SEQ IDNO: 24. In several embodiments, the chimeric receptor comprises afragment of NKG2D coupled to NCR1. In several embodiments, such achimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO:27. In several embodiments, the chimeric receptor comprises the aminoacid sequence of SEQ ID NO: 28.

As discussed above, in several embodiments, the NKG2D fragment iscoupled to NCR2, and the resultant chimeric receptor comprises at leasta portion of the amino acid sequence of SEQ ID NO: 21. Severalembodiments provide for a chimeric receptor comprising a fragment ofNKG2D coupled to NCR3. In several embodiments, the chimeric receptor isencoded by the nucleic acid sequence of SEQ ID NO. 29, and the chimericreceptor comprises the amino acid sequence of SEQ ID NO. 30.

As discussed in more detail below, combinations of transmembrane andintracellular domains are used in several embodiments and provide forsynergistic interactions between the components of the chimeric receptorand yield enhanced cytotoxic effects. In several embodiments, thechimeric receptor comprises the fragment of NKG2D coupled to a CD16transmembrane/intracellular domain and 4-1BB. In several embodiments,the chimeric receptor comprises the fragment of NKG2D coupled to a CD8ahinge, a CD16 transmembrane/intracellular domain and 4-1BB. In severalembodiments, such a chimeric receptor is encoded by the nucleic acidsequence of SEQ ID NO: 25. In several embodiments, the resultantchimeric receptor comprises the amino acid sequence of SEQ ID NO: 26.

In several embodiments, NCR1 is used in conjunction with the NKG2Dfragment. In several embodiments, the NKG2D fragment is linked to NCR1alone. In additional embodiments, the chimeric receptor comprises thefragment of NKG2D coupled to NCR1 and 4-1BB. In some such embodiments,the chimeric receptor comprises the NCR1 amino acid sequence of SEQ IDNO: 20.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to CD8a, 4-1BB and CD3z. In several embodiments, such anNKG2D/CD8a/4-1bb/CD3z chimeric receptor is encoded by the nucleic acidsequence of SEQ ID NO. 18. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO. 19.

In several embodiments, NCR3 is included in the chimeric receptor. Forexample, an NKG2D/NCR3 construct is provided for in several embodiments.The resultant chimeric receptor thereby comprises the NCR3 amino acidsequence of SEQ ID NO: 22. In several embodiments, the chimeric receptorcomprises a NKG2D/NCR2/4-1BB construct or an NKG2D/NCR3/4-1BB construct.

In several embodiments, linkers, hinges, or other “spacing” elements areprovided for in the chimeric receptor constructs. For example, inseveral embodiments, the effector domain comprises a linker. In severalembodiments, the polynucleotides encode a GS linker between the portionsof the construct, such as between any of 4-1BB, CD16, NCR1, NCR3, 2B4 orNKp80. In several embodiments, one or more GS linkers are provided for,for example, 1, 2, 3, 4, 5, 6, or more. In several embodiments, there isprovided for a chimeric receptor comprising a hinge region. Depending onthe location within a particular construct, a hinge region can besynonymous with a linker region, and vice versa. In several embodiments,the hinge region is encoded by the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the hinge region can be truncated to a desiredlength and is therefore encoded by a fragment of the nucleic acidsequence of SEQ ID NO: 5. In several embodiments, a glycine-serine motifis used as a hinge. In several embodiments, the hinge region iscomprises a glycine-serine repeating motif having the amino acidsequence of (GGGGS)n (SEQ ID NO: 31) where n is the number of repeats.In several embodiments, 9 repeats are used, resulting in a hinge regioncomprising the amino acid sequence of SEQ ID NO: 33. In severalembodiments, 3 repeats are used, resulting in a hinge region comprisingthe amino acid sequence of SEQ ID NO: 34.

In several embodiments, two separate molecules can be used as a hinge orlinker, such as the amino acid sequence of SEQ ID NO: 32 (CD8a/GS3). Inseveral embodiments, portions of a beta adrenergic receptor are used asa hinge or linker. In several embodiments, portions of the beta-2adrenergic receptor are used. In one embodiment, an extracellular domainof the beta-2 adrenergic receptor is used, which is encoded by thenucleic acid sequence of SEQ ID NO: 40. In some embodiments, the firsttransmembrane helix of the beta-2 adrenergic receptor is used, which isencoded by the nucleic acid sequence of SEQ ID NO: 42. Depending on theembodiment, these two beta-2 adrenergic receptor portions are usedtogether in the chimeric receptor. In several embodiments, theextracellular receptor domain further comprises a CD8a signal peptide,wherein the signal peptide comprises the nucleic acid sequence of SEQ IDNO. 4. Other signal peptides are optionally used, depending on theembodiment. Signal peptides may be employed in a multimeric format,according to some embodiments.

In several embodiments, the effector domain comprises one or morehemi-ITAM sequences. In some such embodiments, the hemi-ITAM comprisesthe amino acid motif DGYXXL (where X is any amino acid; SEQ ID NO: 14).Multiple hemi-ITAMs are used in some embodiments. In severalembodiments, the hemi-ITAM comprises NKp80. In several embodiments, theeffector domain comprises one or more ITSM sequences. ITSM sequences areused in conjunction with hemi-ITAM motifs in several embodiments. Inseveral embodiments, the ITSM comprises the amino acid motif S/TXYXXL/I(where X is any amino acid; SEQ ID NO. 15). In several embodiments, theeffector comprises a 2B4 domain.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D coupled to a GS3 linker, a CD8a hinge, a CD16transmembrane/intracellular domain and 4-1BB. In several embodiments,the chimeric receptor comprises a fragment of NKG2D coupled to a GS3linker, a CD16 transmembrane/intracellular domain and 4-1BB. In severalembodiments, the chimeric receptor comprises a fragment of NKG2D coupledto a CD16 transmembrane/intracellular domain and 4-1BB. In severalembodiments, the chimeric receptor comprises a fragment of NKG2D coupledto a CD8a hinge, a CD8a transmembrane domain, 4-1BB, and 2B4. In severalembodiments, the chimeric receptor comprises a fragment of NKG2D coupledto a beta-adrenergic extracellular domain, a beta-adrenergictransmembrane domain, 4-1BB, and 2B4. In several embodiments, thechimeric receptor comprises a fragment of NKG2D coupled to a CD8a hinge,a CD8a transmembrane domain, 4-1BB, 2B4, a GS3 linker, and NKp80. Inseveral embodiments, the chimeric receptor comprises a fragment of NKG2Dcoupled to a CD8a hinge, a CD8a transmembrane domain, 4-1BB, a GS3linker, and NKp80. In several embodiments, the chimeric receptorcomprises a fragment of NKG2D, wherein the fragment is encoded by asequence that is codon optimized coupled to a GS3 linker, an additionalNKG2D fragment, a beta-adrenergic extracellular domain, abeta-adrenergic transmembrane domain, 4-1BB, an additional GS3 linker,and NKp80. In several embodiments, the chimeric receptor comprises afragment of NKG2D that is codon optimized coupled to a GS3 linker, anadditional NKG2D fragment, a CD8a hinge, a CD8a transmembrane domain,4-1BB, an additional GS3 linker, and NKp80. In several embodiments, thechimeric receptor comprises a fragment of NKG2D that is codon optimizedcoupled to a GS3 linker, an additional NKG2D fragment, a CD8a hinge, aCD16 transmembrane/intracellular domain, and 4-1BB. In severalembodiments, chimeric receptor comprises a fragment of NKG2D coupled toa CD8a hinge, a CD16 transmembrane/intracellular domain, 4-1BB, and 2B4.In several embodiments, the chimeric receptor comprises a fragment ofNKG2D coupled to a CD8a hinge, a CD16 transmembrane/intracellulardomain, 4-1BB, a GS3 linker, and NKp80. In several embodiments, thechimeric receptor comprises a fragment of NKG2D that is coupled to aCD8a hinge and a CD8a transmembrane domain. In several embodiments, theeffector comprises 4-1BB. In some such embodiments the effectorcomprises 4-1BB optionally in conjunction with one or more of NKp80,2B4, CD3zeta, Dap10, Dap12, CD28, or other signaling domains providedfor herein). In several embodiments, the effector domain furthercomprises CD3zeta. In several embodiments, the effector domain comprisesan intracellular domain of 2B4. In several embodiments, the effectordomain further comprises an intracellular domain of DAP10.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D coupled to a CD8a hinge, a CD8a transmembrane domain, 4-1BB, 2B4,and CD3zeta. In several embodiments, the chimeric receptor is encoded bythe nucleic acid sequence of SEQ ID NO: 58. In several embodiments, thechimeric receptor comprises the amino acid sequence of SEQ ID NO: 59.

Additionally, any of chimeric receptors disclosed herein can also beco-expressed with membrane-bound interleukin 15 (mbIL15). For example,provided for in several embodiments is a polynucleotide encoding achimeric receptor comprising an extracellular receptor domain, whereinthe extracellular receptor domain comprises a peptide that binds nativeligands of NKG2D, wherein the peptide that binds native ligands of NKG2Dis a fragment of NKG2D, a transmembrane region, an effector domain, thepolynucleotide being co-expressed with an additional construct encodingmembrane-bound interleukin 15 (mbIL15). In several embodiments, chimericreceptors as discussed herein are co-expressed with mblL-15. In severalembodiments, the effector domain comprises 4-1BB and CD3 zeta, and thetransmembrane region comprises CD8a.

In several embodiments, the chimeric receptors are engineered such thatthey do not include DNAX-activating protein 10 (DAP10). Additionally, inseveral embodiments, the chimeric receptors are engineered such thatthey do not include an ITAM motif.

In several embodiments, there is provided a polynucleotide encoding achimeric receptor comprising, one, two, or all of: (a) an extracellularreceptor domain comprising a fragment of NKG2D that binds native ligandsof NKG2D, (b) a transmembrane region, wherein the transmembrane regioncomprises CD8a, and (c) an effector domain, wherein the effector domaincomprises 4-1BB and the intracellular domain of 2B4 or DAP10. In severalembodiments, the effector domain comprises 2B4 followed by 4-1BB. Inadditional embodiments, the effector domain comprises 4-1BB followed by2B4. In several embodiments, the effector domain comprises DAP10followed by 4-1BB. In additional embodiments, the effector domaincomprises 4-1BB followed by DAP10. In several embodiments, the chimericreceptor comprises the fragment of NKG2D coupled to a CD8a hinge, a CD8atransmembrane domain, 4-1BB, and DAP10. In several embodiments, thechimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO:60. In several embodiments, the chimeric receptor comprises the aminoacid sequence of SEQ ID NO: 61. In several embodiments, the chimericreceptor comprises the fragment of NKG2D coupled to a CD8a hinge, a CD8atransmembrane domain, 4-1BB, 2B4, and DAP10. In several embodiments, theeffector domain comprises 4-1BB, followed by DAP10, followed by 2B4. Inseveral embodiments, the chimeric receptor is encoded by the nucleicacid sequence of SEQ ID NO: 62. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 63. In severalembodiments, the effector domain comprises 4-1BB, followed by 2B4,followed by DAP10. In several embodiments, the chimeric receptor isencoded by the nucleic acid sequence of SEQ ID NO: 64. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 65.

In several embodiments, the chimeric receptor comprises acodon-optimized fragment of NKG2D coupled to an intracellular effectordomain. In several embodiments, multiple fragments of NKG2D areemployed, for example, an additional NKG2D fragment (optionally codonoptimized) is coupled to the first fragment by, for example, a GS3linker. In several embodiments, such chimeric receptors further comprisea CD8a hinge, a CD8a transmembrane domain, 4-1BB, and CD3zeta. Inseveral embodiments, the chimeric receptor is encoded by the nucleicacid sequence of SEQ ID NO: 66. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 67. In severalembodiments, the polynucleotide is co-expressed with an additionalconstruct encoding membrane-bound interleukin 15 (mbIL15).

In several embodiments, there is provided a polynucleotide encoding achimeric receptor comprising an extracellular receptor domain,comprising a fragment of NKG2D that binds a native ligand of NKG2D andis encoded by a fragment of SEQ ID NO: 1, a transmembrane regioncomprising a CD3zeta transmembrane region, and an effector domain. Inseveral embodiments, there is provided a polynucleotide encoding achimeric receptor comprising an extracellular receptor domain,comprising a fragment of NKG2D that binds a native ligand of NKG2D andis encoded by SEQ ID NO. 2, a transmembrane region comprising a CD3zetatransmembrane region, and an effector domain. In several embodiments,there is provided a polynucleotide encoding a chimeric receptorcomprising an extracellular receptor domain, comprising a fragment ofNKG2D that binds a native ligand of NKG2D and is encoded by SEQ ID NO.3, a transmembrane region comprising a CD3zeta transmembrane region, andan effector domain. In several embodiments, there is provided apolynucleotide encoding a chimeric receptor comprising an extracellularreceptor domain, comprising a fragment of NKG2D that binds a nativeligand of NKG2D and is encoded by SEQ ID NO. 68, a transmembrane regioncomprising a CD3zeta transmembrane region, and an effector domain. Inseveral embodiments, fragments of the NKG2D encoded by any of SEQ ID NO.2, 3, or 68 may also be used. In several embodiments, the CD3zetatransmembrane region comprises the amino acid sequence of SEQ ID NO: 69.Fragments of the sequence of SEQ ID NO: 69 are also use, in severalembodiments, the fragments retaining the ability to transduce at leastabout 65%, about 75%, about 85%, or about 95% of the signal transductionof a native CD3 zeta subunit (including dimers). In several embodiments,the extracellular receptor domain further comprises additional residesadjacent to the CD3zeta transmembrane region. In several embodiments,the additional amino acids are extracellular residues of a nativeCD3zeta sequence. In other embodiments, the additional amino acids arerandomly selected. In several embodiments, there are 2, 3, 4, 5, 6, 8,10, 15, or 20 additional amino acids. In several embodiments, thechimeric receptor domain comprises a hinge region, which in severalembodiments, a CD8a hinge encoded by the nucleic acid sequence of SEQ IDNO: 5. In several embodiments, the hinge region is a CD8a hinge encodedby a fragment of the nucleic acid sequence of SEQ ID NO: 5. Depending onthe embodiment, the fragment is about 75%, about 80%, about 85%, about90%, about 95% of the length of the nucleic acid sequence of SEQ ID NO:5. Depending on the embodiment, the fragment is about 75%, about 80%,about 85%, about 90%, about 95%, about 98%, or about 99% homologous tothe nucleic acid sequence of SEQ ID NO: 5. In several embodiments, theextracellular receptor domain further comprises a CD8a signal peptide,which, depending on the embodiment, can comprise the nucleic acidsequence of SEQ ID NO. 4. In several embodiments, the effector domaincomprises 4-1BB. In several embodiments, the effector domain comprises aCD16 intracellular domain. In several embodiments, the effector domaincomprises 4-1BB and CD16 (with either moiety being “first” vs. “second”in the construct). In several embodiments, repeats of one or more of4-1BB and/or CD16 are used.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D that is codon optimized and is coupled to a CD8a hinge, a CD3zetatransmembrane region, and an effector domain comprising 4-1BB. Inseveral embodiments, the chimeric receptor is encoded by the nucleicacid sequence of SEQ ID NO: 78. In several embodiments, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 79.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D that is codon optimized coupled to a CD8a hinge, a CD3zetatransmembrane region, and an effector domain comprising CD16 followed by4-1BB. In several embodiments, the chimeric receptor comprises the aminoacid sequence of SEQ ID NO: 71. In several embodiments, the chimericreceptor is encoded by the nucleic acid sequence of SEQ ID NO: 70.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D that is codon optimized and coupled to a CD8a hinge, a CD3zetatransmembrane region, and an effector domain comprising 4-1BB followedby CD16, optionally coupled by a GS3 linker. In several embodiments, thechimeric receptor comprises the amino acid sequence of SEQ ID NO: 85. Inseveral embodiments, the chimeric receptor is encoded by the nucleicacid sequence of SEQ ID NO: 84.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D that is codon optimized and is coupled to a GS3 linker, anadditional NKG2D fragment, a CD8a hinge, a CD3zeta transmembrane region,and an effector domain comprising a CD16 and 4-1BB. In severalembodiments, the chimeric receptor is encoded by the nucleic acidsequence of SEQ ID NO: 72. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 73.

In several embodiments, the effector domain includes NKp80. In severalembodiments, the effector domain is NKp80. In several embodiments, thechimeric receptor comprises a fragment of NKG2D that is coupled to aCD8a hinge, a CD3zeta transmembrane region, and an effector domaincomprising a CD16, 4-1BB, and NKp80, and optionally including a GS3linker. In several embodiments, the chimeric receptor is encoded by thenucleic acid sequence of SEQ ID NO: 74. In several embodiments, thechimeric receptor comprises the amino acid sequence of SEQ ID NO: 75. Inseveral embodiments, the chimeric receptor comprises the fragment ofNKG2D that is codon optimized and is coupled to a GS3 linker, anadditional NKG2D fragment (optionally codon optimized), a CD8a hinge, aCD3zeta transmembrane region, and an effector domain comprising a CD16,4-1BB, and NKp80, and optionally including a GS3 linker. In severalembodiments, the chimeric receptor is encoded by the nucleic acidsequence of SEQ ID NO: 76. In several embodiments, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 77. In severalembodiments, the chimeric receptor comprises a fragment of NKG2D that iscodon optimized and is coupled to a CD8a hinge, a CD3zeta transmembraneregion, and an effector domain comprising 4-1BB and NKp80, andoptionally including a GS3 linker. In several embodiments, the chimericreceptor is encoded by the nucleic acid sequence of SEQ ID NO: 82. Inseveral embodiments, the chimeric receptor comprises the amino acidsequence of SEQ ID NO: 83.

In several embodiments, the effector domain comprises CD3zeta. Inseveral embodiments, the chimeric receptor comprises a fragment of NKG2Dthat is codon optimized and is coupled to a CD8a hinge, a CD3zetatransmembrane region, and an effector domain comprising 4-1BB andCD3zeta. In several embodiments, the chimeric receptor is encoded by thenucleic acid sequence of SEQ ID NO: 80. In several embodiments, thechimeric receptor comprises the amino acid sequence of SEQ ID NO: 81.

In several embodiments, the effector domain comprises FcRγ. In severalembodiments, the chimeric receptor comprises a fragment of NKG2D coupledto a CD8a hinge, a CD3zeta transmembrane region, and an effector domaincomprising 4-1BB and FcRγ. In several embodiments, the chimeric receptoris encoded by the nucleic acid sequence of SEQ ID NO: 86. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 87.

In several embodiments, the effector domain comprises CD28. In severalembodiments, the chimeric receptor comprises a fragment of NKG2D coupledto a CD8a hinge, a CD3zeta transmembrane region, and an effector domaincomprising CD28 and CD3zeta. In several embodiments, the chimericreceptor is encoded by the nucleic acid sequence of SEQ ID NO: 102. Inseveral embodiments, the chimeric receptor comprises the amino acidsequence of SEQ ID NO: 103.

In several embodiments, the effector domain comprises a GS linker.

In several embodiments, the polynucleotides disclosed herein areco-expressed with membrane-bound interleukin 15 (mbIL15).

In several embodiments, a polynucleotide encoding a chimeric receptorcomprising an extracellular receptor domain comprising a fragment ofNKG2D that is capable of binding a native ligand of NKG2D and is encodedby a fragment of any one of the sequence of SEQ ID NO: 1, of SEQ ID NO.2, of SEQ ID NO. 3, or SEQ ID NO. 68, and an effector domain comprisinga transmembrane region and an intracellular signaling domain. In severalembodiments, there is provided a polynucleotide encoding a chimericreceptor comprising an extracellular receptor domain comprising afragment of NKG2D that is capable of binding a native ligand of NKG2Dand is encoded by (i) a fragment of the sequence of SEQ ID NO: 1, (ii)the sequence of SEQ ID NO. 2, (iii) the sequence of SEQ ID NO. 3, or(iv) the sequence of SEQ ID NO. 68, and an effector domain comprising atransmembrane region and an intracellular signaling domain. In severalembodiments, a polynucleotide encoding a chimeric receptor comprising anextracellular receptor domain comprising a fragment of NKG2D that iscapable of binding a native ligand of NKG2D and is encoded by thesequence of SEQ ID NO. 2, and an effector domain comprising atransmembrane region and an intracellular signaling domain. In severalembodiments, a polynucleotide encoding a chimeric receptor comprising anextracellular receptor domain comprising a fragment of NKG2D that iscapable of binding a native ligand of NKG2D and is encoded by thesequence of SEQ ID NO. 3, and an effector domain comprising atransmembrane region and an intracellular signaling domain. In severalembodiments, a polynucleotide encoding a chimeric receptor comprising anextracellular receptor domain comprising a fragment of NKG2D that iscapable of binding a native ligand of NKG2D and is encoded by a fragmentof the sequence of SEQ ID NO. 68, and an effector domain comprising atransmembrane region and an intracellular signaling domain. In severalembodiments, the extracellular receptor domain comprises a hinge region.In several embodiments, the hinge region is a CD8a hinge encoded by thenucleic acid sequence of SEQ ID NO: 5, or optionally a fragment of thenucleic acid sequence of SEQ ID NO: 5 (e.g., a fragment having about75%, about 85%, about 95% homology to SEQ ID NO: 5). In severalembodiments, the hinge region is an Immunoglobulin G4 (IgG4) hingeencoded by the nucleic acid sequence of SEQ ID NO: 104. In severalembodiments, the hinge region is an Immunoglobulin G4 (IgG4) hingeencoded by a fragment of the nucleic acid sequence of SEQ ID NO: 104(e.g., a fragment having about 75%, about 85%, about 95% homology to SEQID NO: 104). In several embodiments, the extracellular receptor domainfurther comprises a CD8a signal peptide, wherein the signal peptidecomprises the nucleic acid sequence of SEQ ID NO. 4. In severalembodiments, the effector domain comprises at least one signalingdomains selected from the group consisting of OX40 (CD134), CD3zeta,4-1BB, CD28 and DAP12. In several embodiments, the chimeric receptortransmembrane domain comprises a CD8 transmembrane domain. In severalembodiments, the chimeric receptor comprises IL-15 linked (optionally bya GS3 linker) to the fragment of NKG2D coupled to a CD8a hinge, a CD8atransmembrane domain, 4-1BB, and CD3z. In several embodiments, thechimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO:88. In several embodiments, the chimeric receptor comprises the aminoacid sequence of SEQ ID NO: 89.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D coupled to an IgG4 hinge, a CD8a transmembrane domain, 4-1BB, andCD3zeta. In several embodiments, the chimeric receptor is encoded by thenucleic acid sequence of SEQ ID NO: 96. In several embodiments, thechimeric receptor comprises the amino acid sequence of SEQ ID NO: 97.

In several embodiments, the effector domain comprises OX40. In severalembodiments, the chimeric receptor comprises the fragment of NKG2Dcoupled to a CD8a hinge, a CD8a transmembrane domain, OX40, and CD3z. Inseveral embodiments, the chimeric receptor is encoded by the nucleicacid sequence of SEQ ID NO: 90. In several embodiments, the chimericreceptor is encoded by the nucleic acid sequence of SEQ ID NO: 109. Inseveral embodiments, the chimeric receptor comprises the amino acidsequence of SEQ ID NO: 91. In several embodiments, the chimeric receptorcomprises a fragment of NKG2D coupled to an IgG4 hinge, a CD8atransmembrane domain, OX40 and CD3zeta. In several embodiments, thechimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO:100. In several embodiments, the chimeric receptor comprises the aminoacid sequence of SEQ ID NO: 101.

In several embodiments, the chimeric receptor comprises a CD28transmembrane/intracellular domain. In several embodiments, the chimericreceptor comprises the fragment of NKG2D coupled to a CD8a hinge, a CD28transmembrane/intracellular domain, and CD3zeta. In several embodiments,the chimeric receptor is encoded by the nucleic acid sequence of SEQ IDNO: 92. In several embodiments, the chimeric receptor comprises theamino acid sequence of SEQ ID NO: 93.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D coupled to a CD8a hinge, a CD28 transmembrane/intracellulardomain, 4-1BB, and CD3zeta. In several embodiments, the chimericreceptor is encoded by the nucleic acid sequence of SEQ ID NO: 94. Inseveral embodiments, the chimeric receptor comprises the amino acidsequence of SEQ ID NO: 95.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D coupled to an IgG4 hinge, a CD28 transmembrane/intracellulardomain and CD3zeta. In several embodiments, the chimeric receptor isencoded by the nucleic acid sequence of SEQ ID NO: 98. In severalembodiments, the chimeric receptor comprises the amino acid sequence ofSEQ ID NO: 99.

In several embodiments, the effector domain comprises a GS linker. Inseveral embodiments, the polynucleotides disclosed herein are configuredto be co-expressed (either on the same polynucleotide, or anotherpolynucleotide) with membrane-bound interleukin 15 (mbIL15).

Any of the chimeric receptors can optionally include an extracellularreceptor domain that includes a second peptide that binds native ligandsof NKG2D. In several embodiments, the second peptide is homologous withNKG2D, while in other embodiments, the second peptide is heterologouswith respect to the NKG2D. Whether the chimeric receptor includes adimerized extracellular receptor domain, the extracellular receptordomains can recognize at least the following native ligands of NKG2D:MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 or ULBP6.

As discussed in more detail below, functional variants of the NKG2Dligand binding domains are employed in several embodiments. For examplethe peptide that binds native ligands of NKG2D has, in severalembodiments, at least 80% homology to SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, or SEQ ID NO: 68. In several embodiments, the peptide that bindsnative ligands of NKG2D has at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% homology to SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 68.

Additionally provided for herein in several embodiments are vectors forexpressing the chimeric receptors. In several embodiments, thepolynucleotides provided for herein are mRNA and can include an operablelinkage to least one regulatory element for the expression of thechimeric receptor. In several embodiments, the polynucleotides furtherinclude one or more internal ribosome entry site (IRES). In severalembodiments, the vector is a retrovirus.

Engineered natural killer cells are also provided for, in severalembodiments, that express any of the chimeric receptor constructsdisclosed herein, the engineered NK cells exhibiting enhanced cytotoxiceffects against target cells. Enhanced cytotoxic effects include, butare not limited to, higher affinity for target (e.g., cancerous) cellsas compared to normal (e.g., non-cancerous) cells, a greater killingeffect directed against target cells, reduced off-target effects,increased duration of cytotoxic effects, more efficient cytotoxicity,and the like. Such enhanced effects can be identified through the use ofvarious in vitro cytotoxicity assays (e.g., measurement of cytokineproduction, etc.), measurement of target cell death, or through variousclinical outcomes (e.g., reduction in tumor burden). In severalembodiments, the engineered NK cells are an autologous cell isolatedfrom a patient. In additional embodiments, the engineered NK cells aregenerated from allogeneic cells isolated from a donor. Such engineeredNK cells as disclosed herein are used, in several embodiments, toenhance NK cell cytotoxicity in a mammal in need thereof, byadministering the NK cells. These engineered NK cells are used, inseveral embodiments for treating or preventing cancer or an infectiousdisease in a mammal. The polynucleotides encoding, the vectors carrying,and the NK cells expressing the various chimeric receptors disclosedherein can also be used, in several embodiments in the manufacture of amedicament for enhancing NK cell cytotoxicity (e.g., to treat or preventcancer or an infectious disease). In several embodiments, the chimericreceptor constructs disclosed herein do not significantly increase thecytotoxicity of the engineered NK cells against normal cells and, asdescribed herein, are advantageously improved as compared tonon-engineered NK cells. In several embodiments, there is provided apolynucleotide encoding a chimeric receptor comprising an extracellularreceptor domain, a transmembrane region, and an effector domain. Inseveral embodiments, the extracellular receptor domain comprises apeptide that binds native ligands of Natural Killer Group 2 member D(NKG2D), wherein the peptide that binds native ligands of NKG2D is afragment of NKG2D. Several embodiments, relate to a polynucleotideencoding a chimeric receptor comprising: (a) an extracellular receptordomain, wherein said extracellular receptor domain comprises a peptidethat binds native ligands of Natural Killer Group 2 member D (NKG2D),wherein the peptide that binds native ligands of NKG2D is a fragment ofNKG2D, wherein the fragment of NKG2D is encoded by a polynucleotidecomprising: (i) a fragment of the sequence of SEQ ID NO: 1, (ii) thesequence of SEQ ID NO. 2, (iii) the sequence of SEQ ID NO. 3, or (iv)the sequence of SEQ ID NO. 68, (b) a transmembrane region, and (c) aneffector domain.

In several embodiments, there is provided a polynucleotide encoding achimeric receptor comprising: (a) an extracellular receptor domain,wherein said extracellular receptor domain comprises a peptide thatbinds native ligands of Natural Killer Group 2 member D (NKG2D), whereinthe peptide that binds native ligands of NKG2D is a fragment of NKG2D,wherein the fragment of NKG2D is encoded by a polynucleotide comprising:(i) a fragment of the sequence of SEQ ID NO: 1, (ii) the sequence of SEQID NO. 2, (iii) the sequence of SEQ ID NO. 3, (iv) or the sequence ofSEQ ID NO. 68; and (b) an effector domain comprising a transmembraneregion and an intracellular signaling domain.

In several embodiments, the transmembrane region comprises a CD3zetatransmembrane region. In several embodiments, the CD3zeta transmembraneregion comprises the amino acid sequence of SEQ ID NO: 69. In severalembodiments, the transmembrane region comprises CD8a. In severalembodiments, the effector domain comprises 4-1BB, an intracellulardomain of 2B4, NKp80, a CD16 intracellular domain, Natural CytotoxicityTriggering Receptor 1 (NCR1), Natural Cytotoxicity Triggering Receptor 2(NCR2), Natural Cytotoxicity Triggering Receptor 3 (NCR3), and/or anintracellular domain of DAP10. In one embodiment, the effector domaincomprises 4-1BB and CD16. In several embodiments, the effector domaincomprises 4-1BB and CD3 zeta. In several embodiments, the effectordomain comprises 4-1BB and an intracellular domain of 2B4 or DAP10. Inseveral embodiments, the effector domain comprises 2B4 followed by 4-1BBwhile in other embodiments the effector domain comprises 4-1BB followedby 2B4. In several embodiments, the effector domain comprises DAP10followed by 4-1BB. In several embodiments, the effector domain comprises4-1BB followed by DAP10. In several embodiments, the effector domainfurther comprises CD3zeta. In several embodiments, the effector domaincomprises at least one signaling domain selected from the groupconsisting of OX40 (CD134), CD3zeta, 4-1BB, CD28 and DAP12. In severalembodiments the effector domain comprises one or more hemi-ITAMsequences. In several embodiments, the hemi-ITAM comprises the aminoacid sequence of SEQ ID NO. 14. In several embodiments, the hemi-ITAMcomprises the amino acid sequence of SEQ ID NO. 37. In severalembodiments, the effector domain comprises one or more ITSM sequences.In several embodiments, the ITSM comprises the amino acid sequence ofSEQ ID NO. 15 or the amino acid sequence of SEQ ID NO. 35

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D coupled to a CD8a hinge, a CD8a transmembrane domain, 4-1BB, 2B4,and CD3zeta. In one embodiment, the chimeric receptor is encoded by thenucleic acid sequence of SEQ ID NO: 58. In one embodiment, the chimericreceptor comprises the amino acid sequence of SEQ ID NO: 59. In severalembodiments, the chimeric receptor comprises a fragment of NKG2D coupledto a CD8a hinge, a CD8a transmembrane domain, 4-1BB, and DAP10. Inseveral embodiments, the chimeric receptor is encoded by the nucleicacid sequence of SEQ ID NO: 60 and comprises the amino acid sequence ofSEQ ID NO: 61.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D coupled to a CD8a hinge, a CD8a transmembrane domain, 4-1BB, 2B4,and DAP10. In several embodiments, the effector domain comprises 4-1BB,followed by DAP10, followed by 2B4. In some embodiments, the chimericreceptor is encoded by the nucleic acid sequence of SEQ ID NO: 62 andthe chimeric receptor comprises the amino acid sequence of SEQ ID NO:63. In several embodiments, the effector domain comprises 4-1BB,followed by 2B4, followed by DAP10. In several embodiments, the chimericreceptor is encoded by the nucleic acid sequence of SEQ ID NO: 64 andthe chimeric receptor comprises the amino acid sequence of SEQ ID NO:65.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D that is codon optimized coupled to a GS3 linker, an additionalNKG2D fragment, a CD8a hinge, a CD8a transmembrane domain, 4-1BB, andCD3zeta. In one embodiment, the chimeric receptor is encoded by thenucleic acid sequence of SEQ ID NO: 66. In several embodiments, thechimeric receptor comprises the amino acid sequence of SEQ ID NO: 67.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D that is codon optimized coupled to a CD8a hinge, a CD3zetatransmembrane region, and an effector domain comprising 4-1BB, isencoded by the nucleic acid sequence of SEQ ID NO: 78 and/or comprisesthe amino acid sequence of SEQ ID NO: 79.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D that is codon optimized coupled to a CD8a hinge, a CD3zetatransmembrane region, and an effector domain comprising CD16 followed by4-1BB. In several embodiments, the chimeric receptor is encoded by thenucleic acid sequence of SEQ ID NO: 70 and/or comprises the amino acidsequence of SEQ ID NO: 71.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D that is codon optimized coupled to a CD8a hinge, a CD3zetatransmembrane region, and an effector domain comprising 4-1BB followedby a GS3 linker and CD16. In one embodiment, the chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 85 and/or is encoded bythe nucleic acid sequence of SEQ ID NO: 84.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D that is codon optimized coupled to a GS3 linker, an additionalNKG2D fragment, a CD8a hinge, a CD3zeta transmembrane region, and aneffector domain comprising a CD16 and 4-1BB. In one embodiment, thechimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO:72 and/or comprises the amino acid sequence of SEQ ID NO: 73.

In several embodiments, the chimeric receptor comprises IL-15 linked bya GS3 linker to the fragment of NKG2D coupled to a CD8a hinge, a CD8atransmembrane domain, 4-1BB, and CD3zeta, is encoded by the nucleic acidsequence of SEQ ID NO: 88 and/or comprises the amino acid sequence ofSEQ ID NO: 89.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to a IgG4 hinge, a CD8a transmembrane domain, 4-1BB, andCD3zeta is encoded by the nucleic acid sequence of SEQ ID NO: 96, and/orcomprises the amino acid sequence of SEQ ID NO: 97.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to a CD8a hinge, a CD8a transmembrane domain, OX40, andCD3z,is encoded by the nucleic acid sequence of SEQ ID NO: 90, and/orcomprises the amino acid sequence of SEQ ID NO: 91.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to an IgG4 hinge, a CD8a transmembrane domain, OX40 andCD3zeta, is encoded by the nucleic acid sequence of SEQ ID NO: 100,and/or comprises the amino acid sequence of SEQ ID NO: 101.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to a CD8a hinge, a CD28 transmembrane/intracellulardomain, and CD3zeta, is encoded by the nucleic acid sequence of SEQ IDNO: 92, and/or comprises the amino acid sequence of SEQ ID NO: 93.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to a CD8a hinge, a CD28 transmembrane/intracellulardomain, 4-1BB, and CD3zeta, is encoded by the nucleic acid sequence ofSEQ ID NO: 94, and/or comprises the amino acid sequence of SEQ ID NO:95.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to an IgG4 hinge, a CD28 transmembrane/intracellulardomain and CD3zeta, is encoded by the nucleic acid sequence of SEQ IDNO: 98, and/or comprises the amino acid sequence of SEQ ID NO: 99.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D coupled to a CD8a hinge, a CD3zeta transmembrane region, and aneffector domain comprising a CD16, 4-1BB, a GS3 linker, and NKp80. Inone embodiment, the chimeric receptor is encoded by the nucleic acidsequence of SEQ ID NO: 74 and/or comprises the amino acid sequence ofSEQ ID NO: 75.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D that is codon optimized coupled to a GS3 linker, an additionalNKG2D fragment, a CD8a hinge, a CD3zeta transmembrane region, and aneffector domain comprising a CD16, 4-1BB, a GS3 linker, and NKp80. Inone embodiment, the chimeric receptor is encoded by the nucleic acidsequence of SEQ ID NO: 76 and/or comprises the amino acid sequence ofSEQ ID NO: 77. In several embodiments, the chimeric receptor comprises afragment of NKG2D that is codon optimized coupled to a CD8a hinge, aCD3zeta transmembrane region, and an effector domain comprising 4-1BB, aGS3 linker, and NKp80. In one embodiment, the chimeric receptor isencoded by the nucleic acid sequence of SEQ ID NO: 82 and/or comprisesthe amino acid sequence of SEQ ID NO: 83.

In several embodiments, the chimeric receptor comprises a fragment ofNKG2D that is codon optimized coupled to a CD8a hinge, a CD3zetatransmembrane region, and an effector domain comprising 4-1BB andCD3zeta. In one embodiment, the chimeric receptor is encoded by thenucleic acid sequence of SEQ ID NO: 80 and/or comprises the amino acidsequence of SEQ ID NO: 81.

Depending on the embodiment, the effector domain may also comprise FcRγ.For example, in several embodiments, the chimeric receptor comprises afragment of NKG2D coupled to a CD8a hinge, a CD3zeta transmembraneregion, and an effector domain comprising 4-1BB and FcRγ. In oneembodiment, the chimeric receptor is encoded by the nucleic acidsequence of SEQ ID NO: 86 and/or comprises the amino acid sequence ofSEQ ID NO: 87.

Depending on the embodiment, the effector domain may also comprise CD28.For example, in several embodiments, the chimeric receptor comprises afragment of NKG2D coupled to a CD8a hinge, a CD3zeta transmembraneregion, and an effector domain comprising CD28 and CD3zeta. In severalembodiments, the chimeric receptor is encoded by the nucleic acidsequence of SEQ ID NO: 102 and/or comprises the amino acid sequence ofSEQ ID NO: 103.

In several embodiments, the effector domain comprises a GS linker.

In several embodiments, the extracellular receptor domain furthercomprises a CD8a signal peptide, wherein the signal peptide comprisesthe nucleic acid sequence of SEQ ID NO. 4. In several embodiments, theextracellular receptor domain further comprises 2 extracellular residuesof CD3zeta directly adjacent to the CD3zeta transmembrane region. Inseveral embodiments, the extracellular receptor domain comprises a CD8asignal peptide, wherein the signal peptide comprises the nucleic acidsequence of SEQ ID NO. 4.

In several embodiments, the chimeric receptor comprises one or more GS3linkers. In several embodiments, the chimeric receptor domain comprisesa hinge region. In several embodiments, the hinge region is encoded bythe nucleic acid sequence of SEQ ID NO: 5, while in some embodiments,the hinge region is encoded by a fragment of the nucleic acid sequenceof SEQ ID NO: 5. In several embodiments, the hinge region is a CD8ahinge. In several embodiments, the hinge region comprises aglycine-serine repeating motif having the amino acid sequence of SEQ IDNO: 31. In several embodiments, the hinge region comprises the aminoacid sequence of SEQ ID NO: 32 and in some embodiments, the hinge regioncomprises the amino acid sequence of SEQ ID NO: 33. In additionalembodiments, the hinge region is encoded by the nucleic acid sequence ofSEQ ID NO: 34. In several embodiments, the hinge region comprises aportion of the beta-adrenergic receptor. In some such embodiments, thehinge region is encoded by the nucleic acid sequence of SEQ ID NO: 40.In additional embodiments, the hinge region is encoded by the nucleicacid sequence of SEQ ID NO: 42. In several embodiments, the hinge regionis Immunoglobulin G4 (IgG4) hinge encoded by the nucleic acid sequenceof SEQ ID NO: 104. In several embodiments, the hinge region is aImmunoglobulin G4 (IgG4) hinge encoded by a fragment of the nucleic acidsequence of SEQ ID NO: 104. In several embodiments, the chimericreceptor comprises the fragment of NKG2D coupled to a CD8a hinge and aCD8a transmembrane domain.

In one embodiment, the chimeric receptor comprises the fragment of NKG2Dcoupled to CD16, is encoded by the nucleic acid sequence of SEQ ID NO:23, and/or comprises the amino acid sequence of SEQ ID NO: 24. In oneembodiment, the chimeric receptor comprises the fragment of NKG2Dcoupled to NCR1. In some such embodiments, the chimeric receptor isencoded by the nucleic acid sequence of SEQ ID NO: 27 and/or comprisesthe amino acid sequence of SEQ ID NO: 28. In several embodiments, thechimeric receptor comprises at least a portion of the amino acidsequence of SEQ ID NO: 21. In several embodiments, the chimeric receptorcomprises the fragment of NKG2D coupled to NCR3, in several embodimentsis encoded by the nucleic acid sequence of SEQ ID NO. 29 and/orcomprises the amino acid sequence of SEQ ID NO. 30.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to a CD16 transmembrane/intracellular domain and 4-1BB. Inseveral embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to a CD8a hinge, a CD16 transmembrane/intracellular domainand 4-1BB, is encoded by the nucleic acid sequence of SEQ ID NO: 25,and/or comprises the amino acid sequence of SEQ ID NO: 26.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to NCR1 and 4-1BB, wherein the chimeric receptor comprisesthe NCR1 amino acid sequence of SEQ ID NO: 20.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to CD8a, 4-1BB and CD3z, is encoded by the nucleic acidsequence of SEQ ID NO. 18 and/or comprises the amino acid sequence ofSEQ ID NO. 19.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to NCR3 and 4-1BB, and wherein the NCR3 comprises theamino acid sequence of SEQ ID NO: 22. In one embodiment, the chimericreceptor comprises one or more of the NCR1 transmembrane/intracellulardomain of SEQ ID NO: 20 or the NCR3 transmembrane/intracellular domainof SEQ ID NO: 22.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to a GS3 linker, a CD8a hinge, a CD16transmembrane/intracellular domain and 4-1BB. In several embodiments,the chimeric receptor is encoded by the nucleic acid sequence of SEQ IDNO: 43. In several embodiments, the chimeric receptors comprises thefragment of NKG2D coupled to a GS3 linker, a CD16transmembrane/intracellular domain and 4-1BB. In one embodiment, thechimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO:44.

In several embodiments, the the chimeric receptor comprises the fragmentof NKG2D coupled to a CD16 transmembrane/intracellular domain and 4-1BBand is encoded by the nucleic acid sequence of SEQ ID NO: 45.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to a CD8a hinge, a CD8a transmembrane domain, 4-1BB, and2B4 and is encoded by the nucleic acid sequence of SEQ ID NO: 46.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to a beta-adrenergic extracellular domain, abeta-adrenergic transmembrane domain, 4-1BB, and 2B4 and is encoded bythe nucleic acid sequence of SEQ ID NO: 47.

In several embodiments the chimeric receptor comprises the fragment ofNKG2D coupled to a CD8a hinge, a CD8a transmembrane domain, 4-1BB, 2B4,a GS3 linker, and NKp80 and is encoded by the nucleic acid sequence ofSEQ ID NO: 48.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to a CD8a hinge, a CD8a transmembrane domain, 4-1BB, a GS3linker, and NKp80 and is encoded by the nucleic acid sequence of SEQ IDNO: 49.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D that is codon optimized coupled to a GS3 linker, an additionalNKG2D fragment, a beta-adrenergic extracellular domain, abeta-adrenergic transmembrane domain, 4-1BB, an additional GS3 linker,and NKp80 and is encoded by the nucleic acid sequence of SEQ ID NO: 50.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D that is codon optimized coupled to a GS3 linker, an additionalNKG2D fragment, a CD8a hinge, a CD8a transmembrane domain, 4-1BB, anadditional GS3 linker, and NKp80 and is encoded by the nucleic acidsequence of SEQ ID NO: 51.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D that is codon optimized coupled to a GS3 linker, an additionalNKG2D fragment, a CD8a hinge, a CD16 transmembrane/intracellular domain,and 4-1BB and is encoded by the nucleic acid sequence of SEQ ID NO: 52.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to a CD8a hinge, a CD16 transmembrane/intracellulardomain, 4-1BB, and 2B4 and is encoded by the nucleic acid sequence ofSEQ ID NO: 53.

In several embodiments, the chimeric receptor comprises the fragment ofNKG2D coupled to a CD8a hinge, a CD16 transmembrane/intracellulardomain, 4-1BB, a GS3 linker, and NKp80 and is encoded by the nucleicacid sequence of SEQ ID NO: 54.

In several embodiments, the chimeric receptor constructs are encoded bya polynucleotide that encodes a chimeric receptor wherein theextracellular receptor domain comprises a second peptide that bindsnative ligands of NKG2D, (e.g., one or more of MICA, MICB, ULBP1, ULBP2,ULBP3, ULBP4, ULBP5 or ULBP6. Depending on the embodiment, the peptidethat binds native ligands of NKG2D has at least 80% homology to SEQ IDNO: 1, SEQ ID NO: 2, or SEQ ID NO. 3.

In several embodiments, the polynucleotide is co-expressed with anadditional construct encoding membrane-bound interleukin 15 (mbIL15). Inseveral embodiments, the chimeric receptor is encoded by the nucleicacid sequence of SEQ ID NO: 18. In several embodiments, the chimericreceptor is encoded by the amino acid sequence of SEQ ID NO: 19.

According to several embodiments, the chimeric receptor does notcomprise DNAX-activating protein 10 (DAP10) and/or the chimeric receptordoes not encode an immunoreceptor tyrosine-based activation (ITAM)motif.

In several embodiments, the polynucleotides disclosed herein are mRNA.Additionally, in several embodiments, the polynucleotide disclosedherein are operably linked to at least one regulatory element for theexpression of the chimeric receptor.

Also provided for herein are vectors that comprise the polynucleotidesdisclosed herein. In several embodiments, the polynucleotide isoperatively linked to at least one regulatory element for expression ofthe chimeric receptor. In several embodiments, the vector is aretrovirus.

Also provided for herein are genetically engineered natural killer cellscomprising the any one or more of the polynucleotides disclosed herein.In several embodiments, the natural killer cells are for autologous use,while in some embodiments they are for allogeneic use.

Also provided for herein are methods of enhancing NK cell cytotoxicityin a mammal in need thereof, comprising administering to the mammal NKcells, wherein said NK cells express a chimeric receptor encoded by apolynucleotide disclosed herein.

Additionally, there are provided methods for treating or preventingcancer or an infectious disease in a mammal in need thereof, said methodcomprising administering to said mammal a therapeutically effectiveamount of NK cells, wherein said NK cells express a chimeric receptorencoded by a polynucleotide disclosed herein. As disclosed above, the NKcells can be allogeneic or autologous.

There is provided a use of a polynucleotide as disclosed herein in themanufacture of a medicament for enhancing NK cell cytotoxicity in amammal in need thereof. Further there is provided a use of apolynucleotide in the manufacture of a medicament for treating orpreventing cancer or an infectious disease in a mammal in need thereof.

Also provided is the use of a vector comprising a polynucleotidedisclosed herein in the manufacture of a medicament for enhancing NKcell cytotoxicity in a mammal in need thereof Also provided is the useof a vector comprising a polynucleotide disclosed herein in themanufacture of a medicament for treating or preventing cancer or aninfectious disease in a mammal in need thereof.

Also provided is the use of an isolated genetically engineered naturalkiller cell expressing a chimeric receptor as disclosed herein forenhancing NK cell cytotoxicity in a mammal in need thereof Also providedis the use of an isolated genetically engineered natural killer cellexpressing a chimeric receptor as disclosed herein for treating orpreventing cancer or an infectious disease in a mammal in need thereof.

The compositions and related methods summarized above and set forth infurther detail below describe certain actions taken by a practitioner;however, it should be understood that they can also include theinstruction of those actions by another party. Thus, actions such as“administering a population of NK cells expressing a chimeric receptor”include “instructing the administration of a population of NK cellsexpressing a chimeric receptor.”

BRIEF DESCRIPTION OF THE DRAWINGS

The descriptions of the figures below are related to experiments andresults that represent non-limiting embodiments of the inventionsdisclosed herein.

FIGS. 1A-1C depict schematic representations of the chimeric receptorsaccording to several embodiments disclosed herein. FIG. 1A depictsendogenous NKG2D, FIG. 1B depicts NKG2D-DAP10-CD3ζ, and FIG. 1C depictsNKG2D-41BB-CD3ζ.

FIGS. 2A-2B depict schematic representations of the chimeric receptors,according to several embodiments disclosed herein. FIG. 2A depictsNKG2D-CD16 and FIG. 2B depicts NKG2D-CD16-41BB.

FIGS. 3A-3B depict plasmid maps illustrating the point of insertion ofcertain constructs according to several embodiments into the plasmids,illustrated is a Murine Stem Cell Virus (MSCV) plasmid. FIG. 3A showsgene constructs for NKG2D-DAP10-CD3ζ and NKG2D-41BB-CD3ζ that wereinserted into the EcoRI and NotI restriction sites, with removal theIRES-GFP sequence in the vector. FIG. 3B depicts the plasmids forNKG2D-CD16 and NKG2D-CD16-41BB that were inserted into EcoRI and XhoIrestriction sites located in the multiple cloning site (MCS). IRES-GFPsequence in the vector allows for the tracing of transductionefficiency.

FIGS. 4A-4C depict data related to the expression of NKG2D-DAP10-CD3ζand NKG2D-41BB-CD3ζ in NK cells. FIG. 4A shows flow cytometry dataillustrating the percentage of NKG2D-positive NK cells aftertransduction. FIG. 4B shows a dot plots summarizing the percentage ofNKG2D-positive NK cells. FIG. 4C shows data related to the meanfluorescence intensity (MFI) in different group of NK cells aftertransduction.

FIGS. 5A-5C depict data related to the cytotoxicity of the variousconstructs generated from NK cells from Donor 1, Donor 2, and Donor 3(FIGS. 5A, 5B, and 5C, respectively) against cultured REH cells.

FIGS. 6A-6C depict data related to the cytotoxicity of the variousconstructs generated from NK cells from Donor 1, Donor 2, and Donor 3(FIGS. 6A, 6B, and 6C, respectively) against cultured U-2 OS cells.

FIGS. 7A-7B depict data related to the production of interferon-gamma byNK cells expressing various NKG2D constructs in the presence and absenceof stimulation with REH cells. FIG. 7A depicts the relative amount ofIFNγ in the different groups of NK cells with or without stimulation byREH cells. FIG. 7B depicts levels of IFNγ between different groups of NKcells after stimulation (median values represented).

FIGS. 8A-8C depict data related to the expression of NKG2D-DAP10-CD3ζand NKG2D-CD16 in NK cells. FIG. 8A shows flow cytometry dataillustrating the percentage of NKG2D-positive NK cells aftertransduction. FIG. 8B shows a dot plots summarizing the percentage ofNKG2D-positive NK cells. FIG. 8C shows data related to the meanfluorescence intensity (MFI) in different group of NK cells aftertransduction.

FIGS. 9A-9C depict data related to the cytotoxicity of the variousconstructs generated from NK cells from 3 donors (FIGS. 9A, 9B, and 9C,respectively) against cultured REH cells.

FIGS. 10A-10C depict data related to the cytotoxicity of the variousconstructs generated from NK cells from 3 donors (FIGS. 10A, 10B, and10C, respectively) against cultured U-2 OS cells.

FIG. 11 depicts data related to the production of interferon-gamma by NKcells expressing various NKG2D constructs in the presence and absence ofstimulation with REH cells.

FIGS. 12A-12B depict data related to expression of NKG2D-DAP10-CD3ζ andNKG2D-CD16-41BB in NK cells. FIG. 12A shows flow cytometry dataillustrating the percentage of NKG2D-positive NK cells aftertransduction. FIG. 12B shows a histogram related to relative amount ofsurface expression of the various constructs on NK cells.

FIGS. 13A-13B depict data related to the degree of cytotoxicity ofvarious NKG2d constructs. FIG. 13A depicts the degree of cytotoxicityagainst cultured REH cells. FIG. 13B depicts the degree of cytotoxicityagainst cultured U2OS cells.

FIG. 14 schematically depicts construct maps of several NKG2D constructsaccording to some embodiments disclosed herein.

FIG. 15 schematically depicts construct maps of additional NKG2Dconstructs according to some embodiments disclosed herein.

FIGS. 16A-16C depict data related to the expression of the various NKG2Dconstructs in NK cells. FIG. 16A shows data related to the meanfluorescence intensity (MFI) of the various NKG2D constructs in NKcells. FIG. 16B shows flow cytometry data illustrating the percentage ofNKG2D-positive and CD56-positive NK cells after transduction of variousNKG2D constructs into the NK cells of two donors (505 and 870). FIG. 16Cshows data related to the mean fluorescence intensity (MFI) in NK cellsfrom 2 donors seven days after transduction.

FIG. 17 depicts data related to the cytotoxicity of the various NKG2Dconstructs 14 days post-transduction into NK cells at a 1:1 E:T ratio.

FIGS. 18A-18B depicts data related to the expression of the variousNKG2D constructs following transduction into NK cells. FIG. 18A showsdata related to the mean fluorescence intensity (MFI) in NK cells sevendays after transduction. FIG. 18B shows data related to the fold-changein MFI of the various NKG2D constructs relative to the mock-transducedNK cells.

FIGS. 19A-19B depict data related to the cytotoxicity of the variousNKG2D constructs. FIG. 19A shows data related to the cytotoxicity of thevarious NKG2D constructs transduced into NK cells at a 1:1 E:T ratio.FIG. 19B shows data related to the percent change in cytotoxicity of thevarious NKG2D constructs relative to the mock-transduced NK cells.

FIG. 20 depicts data related to the cytotoxicity of the various NKG2Dconstructs 14 days post-transduction into NK cells at a 1:1 E:T ratio.Prior to analysis NK cells were cultured in media supplemented with 40IU of IL-2/mL.

FIG. 21 depicts data related to the cytotoxicity of the various NKG2Dconstructs 10 days post-transduction into Donor 238 NK cells (with 4days of culturing in media supplemented with 40 IU of IL-2/mL every twodays) against cultured REH cells at 1:1 and 1:2 E:T ratios for twohours.

FIG. 22 schematically depicts construct maps of additional NKG2Dconstructs according to embodiments disclosed herein.

FIGS. 23A-23B depict data related to the persistence of the variousNKG2D constructs generated from NK cells from two different donors(Donor 61 and Donor 103 in FIGS. 23A and 23B, respectively). NK cellswere cultured in media supplemented with 40 IU of IL-2/mL.

FIG. 24 depicts data related to the expression of the various NKG2Dconstructs. NK cells were expanded from peripheral blood mononuclearcells (PBMC) of 4 healthy donors (224, 225, 362 and 363) and transducedwith viruses directing the expression of the indicated constructs. Threedays following transduction, NK cells were stained with a fluorescentlylabelled anti-NKG2D antibody and analyzed using flow cytometry. RelativeNKG2D expression was assessed by mean fluorescence intensity (MFI) oflabeled cells.

FIGS. 25A-25B depict data related to the cytotoxicity of NK cellstransduced with various NKG2D constructs. NK cells were expanded fromPBMC of 4 donors; Eight days after transduction, NK cytotoxicity againstcultured REH and HL60 cells (FIGS. 25A and 25B, respectively) wasmeasured at a 1:1 E:T ratio. NK cells were cultured in mediasupplemented with 40 IU of IL-2/mL prior to analysis.

FIGS. 26A-26C depict data related to the production of interferon-gamma(IFNγ), tumor necrosis factor-alpha (TNFα), and granulocyte-macrophagecolony-stimulating factor (GM-CSF) by NK cells expressing various NKG2Dconstructs after overnight stimulation with REH tumor cells. Eight daysafter transduction with the indicated constructs, 1×10⁵ NK cells werestimulated with 1×10⁵ REH cells in individual wells of a 96-well roundbottom plate; after overnight incubation, supernatants were harvested,and cytokine levels measured against relevant standards using a MesoScale Discovery device. FIG. 26A depicts the accumulated levels of IFNγ,FIG. 26B depicts the levels of TNFα, and FIG. 26C depicts the levels ofGM-CSF in the different groups of NK cells following stimulation. Priorto analysis NK cells were cultured in media supplemented with 40 IU ofIL-2/mL.

FIGS. 27A-27B depict data related to the persistence of NK cells fromtwo donors (donors 224 and 225 in FIGS. 27A and 27B, respectively)expressing the various NKG2D constructs 7, 14, and 21 dayspost-transduction. Prior to analysis NK cells were cultured in mediasupplemented with 40 IU of IL-2/mL.

FIGS. 28A-28B depict data related to the cytotoxicity of NK cellstransduced with the indicated NKG2D constructs. NK cytotoxicity wasmeasured against U2OS cells stably transduced to express Red FluorescentProtein; U2OS cells were cultured with NK cells at a 1:4 and 1:2 E:Tratios (FIGS. 28A and 28B, respectively). Live U2OS cells were countedevery 60 minutes for 72 hours using an Incucyte S3 Live-Cell AnalysisSystem. Prior to analysis NK cells were cultured in media supplementedwith 40 IU of IL-2/mL.

DETAILED DESCRIPTION General

The emergence and persistence of aberrant cells (including virallyinfected and malignant cells) underlying many diseases is enabled by aninsufficient immune response to said aberrant cells. A goal ofimmunotherapy is to initiate or augment the response of the patient'simmune system, for example, to boost the ability of immune cells, suchas Natural Killer (NK) cells to damage, kill, or otherwise inhibitdamaged or diseased cells. One immunotherapy approach is the recombinantexpression of chimeric receptors in immune cells for targetedrecognition and destruction of the aberrant cells. In general, chimericreceptors comprise an extracellular receptor domain that recognizesligands on target cells, an anchoring transmembrane domain, and aneffector domain that transduces activating signals upon ligand binding.Some embodiments disclosed herein utilize chimeric receptors having thatgeneral structure, or having variations in that general structure.Additionally, in several embodiments, the transmembrane domain and theeffector domain are separate peptides fused together. In several otherembodiments, the transmembrane and the effector domain are derived fromthe same peptide. In some such embodiments, the transmembrane andeffector domains comprise a single peptide (e.g., one peptide thatpasses through the membrane and is also poised to initiate a signalingcascade). As discussed in more detail below, truncations, mutations,additional linkers/spacer elements, dimers, and the like are used togenerate chimeric receptor constructs that exhibit a desired degree ofexpression in an immune cell (e.g., an NK cell), induce cytotoxicactivity from the NK cell, balanced with a degree of target avidity thatavoids adverse effects on non-target cells. The recombinant expressionof chimeric receptors as disclosed herein on the surface of immune cellscan redirect the targeting of immune cells to aberrant cells of interestas well as augment the immune activation upon engagement.

NK Cells for Immunotherapy

One immunotherapy approach involves administering to patients T cellsengineered to express chimeric receptors to elicit a positive immuneresponse. However, a drawback of this approach is that it necessitatesthe use of autologous cells to prevent the induction ofgraft-versus-host-disease in the patient. As is provided in severalembodiments disclosed herein, compositions comprising engineered NKcells enjoy several advantages. For example, either autologous ordonor-derived allogeneic cells can be employed with an NK cell approach.Additionally, according to several embodiments, the engineered NK cellsas provided for herein do not significantly increase cytotoxicityagainst normal cells. Further, NK cells have a significant cytotoxiceffect, once activated. In view of this, it is unexpected that theengineered NK cells as provided for herein, are able to further elevatethat cytotoxic effect, thus providing an even more effective means ofselectively killing diseased target cells. Accordingly, in severalembodiments, there is provided a method of treating or preventing canceror an infectious disease, comprising administering a therapeuticallyeffective amount of NK cells expressing the chimeric receptors describedherein. In one embodiment, the NK cells administered are autologouscells. In a further embodiment, the NK cells administered aredonor-derived (allogeneic) cells.

In several embodiments, engagement and activation of a recombinant NKcell (e.g., by binding to a ligand on a target cell) expressing achimeric receptor leads to the direct killing of the stressed and/oraberrant cell (e.g., tumor cells, virally-infected cells, etc.) bycytolysis. Accordingly, in several embodiments, there is provided amethod of enhancing NK cell cytotoxicity, comprising administering NKcells engineered to express the chimeric receptors described herein. Inone embodiment, the NK cells administered are autologous cells. In afurther embodiment, the NK cells are donor-derived (allogenic) cells. Inseveral embodiments, engineered NK cells lead to indirect destruction orinhibition of stressed and/or aberrant cell (e.g., tumor cells,virally-infected cells, etc.).

Ligand Binding Domains

As mentioned above, in several embodiments NK cells recognize anddestroy aberrant cells, including tumor cells and virally-infectedcells. The cytotoxic activity of these innate immune cells is regulatedby the balance of signaling from inhibitory and activating receptors,respectively, that reside on the cell surface. The former bindself-molecules expressed on the surface of healthy cells while thelatter bind ligands expressed on aberrant cells. The increasedengagement of activating receptors relative to inhibitory receptorsleads to NK cell activation and target cell lysis. Natural killer Group2 member D (NKG2D) is an important NK cell activating receptor thatrecognizes a number of ligands expressed on stressed and aberrant cells.The surface expression of various NKG2D ligands is generally low inhealthy cells but is upregulated upon malignant transformation or viralinfection. Non-limiting examples of ligands recognized by NKG2D include,but are not limited to, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5,and ULBP6, as well as other molecules expressed on target cells thatcontrol the cytolytic or cytotoxic function of NK cells.

NKG2D's ability to recognize a plurality of surface markers of cellstress and infection make it a potentially useful component of achimeric receptor-based immunotherapy approach. However, complicatingthe use of NKG2D as a chimeric receptor is its relationship with partnerDAP10. NKG2D is a type II transmembrane glycoprotein that formshomodimers and assembles with two homodimers of DNAX-activating protein10 (DAP10) to yield hexameric complexes on the membrane surface. ThisNKG2D-DAP10 association is necessary for both surface membraneexpression of endogenous NKG2D as well as for transduction of theactivation signal upon ligand binding. In several embodiments, a fulllength NKG2D is used. In one embodiment, full length NKG2D has thenucleic acid sequence of SEQ ID NO. 1. According to several embodimentsdisclosed herein, polynucleotides encoding chimeric receptors areprovided wherein the extracellular receptor domain is a fragment ofNKG2D that lacks its native transmembrane or intracellular domains yetadvantageously retains its ability to bind native ligands of NKG2D, aswell as transduce activation signals upon ligand binding. Thus, inseveral embodiments, the chimeric receptor encoded by the polypeptidesdisclosed herein does not comprise DAP10. In several embodiments, theNKG2D fragment is encoded by SEQ ID NO. 2. In several embodiments, thefragment of NKG2D is at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% homologous with full-length wild-typeNKG2D. In several embodiments, the fragment may have one or moreadditional mutations from SEQ ID NO. 2, but retains, or in someembodiments, has enhanced, ligand-binding function. In severalembodiments, the NKG2D fragment is provided as a dimer, trimer, or otherconcatameric format, such embodiments providing enhanced ligand-bindingactivity. In several embodiments, the sequence encoding the NKG2Dfragment is optionally fully or partially codon optimized. In oneembodiment, a sequence encoding a codon optimized NKG2D fragmentcomprises the sequence of SEQ ID NO. 3. Additionally, in severalembodiments signal peptides are used. The species or sequence of thesignal peptide can vary with the construct. However, in severalembodiments, a signal peptide derived from CD8 is used. In oneembodiment, the signal peptide is from CD8a and has the sequence of SEQID NO. 4. In one embodiment, a sequence encoding a codon optimized NKG2Dfragment comprises the sequence of SEQ ID NO. 68. In severalembodiments, the fragment may have one or more additional mutations fromSEQ ID NO. 68, but retains ligand-binding function. In severalembodiments, the fragment may have one or more additional mutations fromSEQ ID NO. 68, but has improved ligand-binding function.

Transmembrane, Signaling and Combination Domains

As mentioned above, the general chimeric antigen receptor structurecomprises at least one transmembrane domain, linking the ligand bindingdomain to a signaling domain(s). In several embodiments, however, atransmembrane domain can also serve to provide signaling function.

In several embodiments, the NKG2D fragment retains at least a portion ofits normal transmembrane domain. In several embodiments, thetransmembrane domain comprises at least a portion of CD8, which is atransmembrane glycoprotein normally expressed on both T cells and NKcells. In several embodiments, the transmembrane domain comprises CD8α,while in some embodiments CD8β is used. In several embodiments, the“hinge” of CD8α has the sequence of SEQ ID NO. 5. In severalembodiments, the CD8α can be truncated or modified, such that it is atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% homologous with the CD8α having the sequence of SEQ ID NO. 5.In several embodiments, CD8β has the sequence of SEQ ID NO. 6. Inseveral embodiments, the CD8β can be truncated or modified, such that itis at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 95% homologous with the CD8β having the sequence of SEQ ID NO.6. In several embodiments, dimers of CD8α and CD8β are used.

In several embodiments, the transmembrane domain comprises CD16, whichserves as a signaling domain as well. CD16 exists in two isoforms, a andb (also known as Fc gamma receptor IIIa and IIIb, respectively). Thesereceptors normally bind to the Fc portion of IgG antibodies that in turnactivates NK cells. Accordingly, in several embodiments, thetransmembrane domain comprises CD16a, while in some embodiments CD16b isused. In several embodiments, CD16a has the sequence of SEQ ID NO. 7. Inseveral embodiments, the CD16a can be truncated or modified, such thatit is at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95% homologous with the CD16a having the sequence of SEQID NO. 7. In several embodiments, CD16b has the sequence of SEQ ID NO.8. In several embodiments, the CD16b can be truncated or modified, suchthat it is at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% homologous with the CD16b having the sequence ofSEQ ID NO. 8. In several embodiments, dimers of CD16a and CD16b areused. In several embodiments the modifications to the CD16 transmembranedomain comprise additional nucleic acid residues to increase the lengthof the domain. Alternatively, CD16 may be shortened. The modificationsto the length of CD16 advantageously can facilitate enhancedligand-receptor interactions.

In several embodiments, the chimeric receptor comprises the NaturalKiller Receptor 2B4 domain (referred to herein as “2B4”, and also knownas CD244), which serves as a signaling domain as well. 2B4 is expressedon NK cells and regulates non-major histocompatibility complex (MHC)restricted killing through interactions between this receptor and itsligands on target cells. In several embodiments, the transmembranedomain comprises 2B4, while in several embodiments the 2B4 domain is anintracellular signaling domain. In several embodiments, 2B4 has thesequence of SEQ ID NO. 9. In several embodiments, the 2B4 can betruncated or modified, such that it is at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% homologous with the2B4 having the sequence of SEQ ID NO. 9. In several embodiments, 2B4 isused as the sole transmembrane/signaling domain in the construct,however, in several embodiments, 2B4 can be used with one or more otherdomains. For example, combinations of CD16, 4-1BB, and/or 2B4 are usedin some embodiments.

In some embodiments, signaling is achieved through DAP10, as mentionedabove. In several embodiments, the fragment of NKG2D associates withDAP10 to provide pro-cytotoxic signals to the NK cell. In severalembodiments, dimers of DAP10 are used. In several embodiments, thetransmembrane domain comprises DAP10. In several embodiments, DAP10 hasthe sequence of SEQ ID NO. 10. In several embodiments, DAP10 can betruncated or modified, such that it is at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% homologous with theDAP10 having the sequence of SEQ ID NO. 10. Similarly, in someembodiments, DAP12 can be used, as it can also transduce such signals.In several embodiments, DAP12 has the sequence of SEQ ID NO. 11. Inseveral embodiments, DAP12 can be truncated or modified, such that it isat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% homologous with the DAP12 having the sequence of SEQ ID NO.11. In several embodiments, heterodimers of DAP10 and DAP12 are used.

In several embodiments, signaling is provided through 4-1BB (also knownas CD137 and tumor necrosis factor receptor superfamily member 9 (TNFRSF9)). 4-1BB is a co-stimulatory immune checkpoint molecule, typicallyfunctioning as a stimulatory molecule for activated T cells (e.g.,crosslinking of 4-1BB enhances T cell proliferation and cytolyticactivity). However, in several embodiments, the function of 4-1BB isadvantageously used in conjunction with NK cells. In severalembodiments, 4-1BB has the sequence of SEQ ID NO. 12. In severalembodiments, 4-1BB can be truncated or modified, such that it is atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% homologous with the 4-1BB having the sequence of SEQ ID NO.12. In several embodiments, 4-1BB is the sole signaling domain, but asdiscussed above, in several embodiments, 4-1BB functions unexpectedlywell in combination with one or more of the othertransmembrane/signaling domains disclosed herein. For example, inseveral embodiments, CD16 in conjunction with 4-1BB provides synergisticstimulation effects, resulting in particularly effective (e.g.,cytotoxic) NK cells. In several embodiments, DAP10 in conjunction with4-1BB provides synergistic stimulation effects, resulting inparticularly effective (e.g., cytotoxic) NK cells. In severalembodiments, DAP10 in conjunction with 4-1BB and/or 2B4 providessynergistic stimulation effects, resulting in particularly effective(e.g., cytotoxic) NK cells. Other improved characteristics result, inseveral embodiments, such as improved expression, improved persistence,and the like.

In several embodiments, the signaling domain comprises at least aportion of the CD3 T cell receptor complex. The T cell receptor complexcomprises multiple subunits, including the zeta, alpha, beta, gamma,delta, and epsilon subunits. In several embodiments, the NK cellsengineered according to several embodiments disclosed herein comprise atleast one of these subunits (or a fragment thereof). In severalembodiments, the signaling domain comprises the CD3 zeta subunit. Inseveral embodiments, CD3 zeta has the sequence of SEQ ID NO. 13. Inseveral embodiments, CD3 zeta can be truncated or modified, such that itis at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 95% homologous with the CD3 zeta having the sequence of SEQ IDNO. 13. In several embodiments, the CD3 zeta is mutated (e.g., aminoacid mutations, insertions, or deletions) such that the domain no longeris consistent with the canonical immunoreceptor tyrosine-basedactivation motif or ITAM motif. Thus, in several embodiments, the NKcells comprise an engineered receptor that does not contain an ITAMmotif. In some embodiments, the resultant engineered NK cells exhibitparticularly enhanced cytotoxicity against target cells, with limited orreduced adverse side effects. This, in several embodiments, results fromthe synergistic interactions of the various portions of the chimericreceptor that are used in that given embodiment. In several embodiments,CD3zeta in conjunction with 4-1BB provides synergistic stimulationeffects, resulting in particularly effective (e.g., cytotoxic) NK cells.In several embodiments, CD3zeta in conjunction with 2B4 providessynergistic stimulation effects, resulting in particularly effective(e.g., cytotoxic) NK cells. In several embodiments, CD3zeta incombination with 2B4 and 4-1BB provides synergistic stimulation effects,resulting in particularly effective (e.g., cytotoxic) NK cells. Inseveral embodiments, the chimeric receptors leverage the dimerization ofCD3zeta via its transmembrane domain. Thus, in several embodiments, thetransmembrane domain comprises the CD3zeta transmembrane domain (or afragment thereof). In some embodiments, 1, 2, 3, 4, 5, 6 or moreextracellular CD3zeta residues (the “juxta-membrane portion”) aredirectly adjacent to the CD3zeta transmembrane domain. In someembodiments, CD3zeta transmembrane domain has the sequence of SEQ ID NO.69. In several embodiments, the CD3zeta transmembrane domain can betruncated or modified, such that it is at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% homologous with theCD3zeta transmembrane domain having the sequence of SEQ ID NO. 69. Inseveral embodiments the modifications to the CD3zeta transmembranedomain comprise additional nucleic acid residues to increase the lengthof the domain. In several embodiments, the CD3zeta transmembrane domainand CD3zeta juxta-membrane portion recruits full-length CD3zeta moleculeto the synapse. In several embodiments, the recruitment of nativeCD3zeta to the engineered receptor (as compared to a receptor without aCD3zeta transmembrane domain) is increased by about 20%, by about 30%,by about 40% by about 50%, or more, depending on the embodiment. Inseveral embodiments, the CD3zeta transmembrane domain is coupled to aneffector domain comprising one or more of CD16, NCR1, NCR2, NCR3, 4-1BB,NKp80, FcRγ, CD3zeta and 2B4.

In several embodiments, the chimeric receptor comprises a CD28 domain.In several embodiments, the transmembrane domain comprises CD28, whilein several embodiments the CD28 domain is an intracellular signalingdomain, while in several embodiments the CD28 domain is atransmembrane/intracellular signaling domain. In several embodiments,the CD28 transmembrane domain has the sequence of SEQ ID NO. 105. Inseveral embodiments, the CD28 transmembrane domain can be truncated ormodified, such that it is at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% homologous with the CD28 havingthe sequence of SEQ ID NO. 105. In several embodiments, the CD28intracellular signaling domain has the sequence of SEQ ID NO. 106. Inseveral embodiments, the CD28 intracellular signaling domain can betruncated or modified, such that it is at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% homologous with theCD28 having the sequence of SEQ ID NO. 106. In several embodiments, CD28is used as the sole transmembrane/signaling domain in the construct,however, in several embodiments, CD28 can be used with one or more otherdomains. For example, combinations of CD28, OX40, 4-1BB, and/or CD3zetaare used in some embodiments.

In several embodiments, the chimeric receptor comprises an OX40 domain.In several embodiments the OX40 domain is an intracellular signalingdomain. In several embodiments, the OX40 intracellular signaling domainhas the sequence of SEQ ID NO. 107. In several embodiments, the OX40intracellular signaling domain can be truncated or modified, such thatit is at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95% homologous with the OX40 having the sequence of SEQ IDNO. 107. In several embodiments, OX40 is used as the soletransmembrane/signaling domain in the construct, however, in severalembodiments, OX40 can be used with one or more other domains. Forexample, combinations of CD28, OX40, 4-1BB, and/or CD3zeta are used insome embodiments.

In still further embodiments, the signaling portion of the chimericreceptor comprises a portion of an ITAM, for example a hemi-tam. Inseveral embodiments, these portions do not make up the canonical ITAMsequence, but rather comprise a portion that still can convey the signalrequired for NK cell cytotoxicity. In several embodiments, the hemi-tamhas the sequence of SEQ ID NO. 14 (wherein X can be any residue). Inseveral embodiments, the hemi-tam can be truncated or modified, suchthat it is at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% homologous with the hemi-tam having the sequenceof SEQ ID NO. 14. In several embodiments, the chimeric receptorconstruct comprises the hemi-tam of SEQ ID NO. 14. In severalembodiments, multiple hemi-tams can be used, for example in a head totail, tail to head, head to head, or tail to tail configuration. Inseveral embodiments, the presence of at least on hemi-tam confersenhanced signaling and cytotoxicity to the NK cells comprising achimeric receptor employing the at least one hemi-tam. As discussed inmore detail below, in several chimeric receptor comprises NKp80, whichis one non-limiting example of a hemi-tam.

In several embodiments, additional signaling regions are used,including, for example, signaling regions derived from receptors of thesignaling lymphocytic activation molecule (SLAM) family. These receptorsinclude, but are not limited to 2B4 (discussed above). Receptors of theSLAM family share a consensus motif that is tyrosine-based, in theircytoplasmic tails. That motif is S/TxYxxL/I, which are referred to asimmunoreceptor tyrosine-based switch motifs (ITSM) (SEQ ID NO. 15).These receptors transmit activation signals through the SLAM-associatedprotein (SAP, encoded by the gene SH2D1A), which recruits the tyrosinekinase Fyn. Thus, according to several embodiments, the signaling regioncomprise a polypeptide sequence (or the nucleic acid encoding the same)comprising an ITSM motif. In several embodiments, the ITSM motif neednot be fully encoded, but the signaling region is able to transmit anactivation signal through SAP (or another similar pathway). In severalembodiments, the ITSM motif has the sequence of SEQ ID NO. 15 (wherein Xcan be any amino acid residue). In several embodiments, the ITSM motifcan be truncated or modified, such that it is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% homologouswith the ITSM motif having the sequence of SEQ ID NO. 15. In severalembodiments, the ITSM motif comprises the sequence of SEQ ID NO. 15.

In addition to these variations in the NKG2D receptor, the transmembranedomain and signaling domain (and the combination transmembrane/signalingdomains), additional co-activating molecules can be provided, in severalembodiments. For example, in several embodiments, the NK cells areengineered to express membrane-bound interleukin 15 (mbIL15). In suchembodiments, the presence of the mbIL15 on the NK cell function tofurther enhance the cytotoxic effects of the NK cell by synergisticallyenhancing the proliferation and longevity of the NK cells. In severalembodiments, mbIL15 has the nucleic acid sequence of SEQ ID NO. 16. Inseveral embodiments, mbIL15 can be truncated or modified, such that itis at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 95% homologous with the sequence of SEQ ID NO. 16. In severalembodiments, the mbIL15 has the amino acid sequence of SEQ ID NO. 17. Inconjunction with the chimeric receptors disclosed herein, suchembodiments provide particularly effective NK cell compositions fortargeting and destroying particular target cells.

Chimeric Receptor Constructs

In view of the disclosure provided herein, there are a variety ofchimeric receptors that can be generated and expressed in NK cells inorder to target and destroy particular target cells, such as diseased orcancerous cells. Non-limiting examples of such chimeric receptors arediscussed in more detail below.

As discussed above, portions of the T cell receptor complex, inparticular CD3zeta, serve as potent activators of immune signalingcascades. Likewise, the receptor 4-1BB, a tumor necrosis factorsuperfamily member, activates NK cells upon ligand binding. In severalembodiments, these two signaling components act in a synergistic mannerto activate NK cells upon binding of a ligand to the chimeric receptor.Thus, in several embodiments, there are provided polynucleotidesencoding a NKG2D/CD8a/4-1BB/CD3zeta chimeric receptor, which comprisesan NKG2D fragment extracellular receptor domain that binds nativeligands of NKG2D, a CD8 transmembrane region, and an effector domaincomprising the signaling domains of 4-1BB and CD3zeta. In oneembodiment, this chimeric receptor is encoded by the nucleic acidsequence of SEQ ID NO: 18. In one embodiment, this chimeric receptor isencoded by the nucleic acid sequence of SEQ ID NO: 108. In yet anotherembodiment, the NKG2D-CD8a-4-1BB-CD3zeta chimeric receptor comprises theamino acid sequence of SEQ ID NO: 19. In several embodiments, thisconstruct is particularly efficacious when the NK cells concurrentlyexpress mbIL15, the mbIL15 provides a further synergistic effect withrespect to the activation and cytotoxic nature of the NK cells. In someembodiments, the sequence of the chimeric receptor may vary from SEQ IDNO. 18 (such as, for example, SEQ ID NO: 108), but remains, depending onthe embodiment, at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, or at least 95% homologous with SEQ ID NO. 18. In severalembodiments, while the chimeric receptor may vary from SEQ ID NO. 18(such as, for example, SEQ ID NO: 108), the chimeric receptor retains,or in some embodiments, has enhanced, NK cell activating and/orcytotoxic function.

The receptor 2B4 possesses several immunoreceptor tyrosine-based switchmotifs (ITSMs) and has the potential to transduce activating signals.Likewise, signaling through the receptor 4-1BB, a tumor necrosis factorsuperfamily member, also activates NK cells upon ligand binding. Thus,capitalizing on the ability of these signaling molecules to cooperate togenerate unexpectedly effectively cytotoxic NK cells, in severalembodiments, there are provided polynucleotides encoding aNKG2D/CD8a/2B4/4-1BB chimeric receptor, which comprises an NKG2Dfragment extracellular receptor domain that binds native ligands ofNKG2D, a CD8a transmembrane region, and an effector domain comprisingthe signaling domains of 4-1BB and 2B4. Additionally, in severalembodiments, this construct can optionally be co-expressed with mbIL15.

In several embodiments, combinations of 2B4 with CD3zeta are used withNK cells to generate enhanced cytotoxicity against target cells. Thus,in several embodiments, there are provided polynucleotides encoding aNKG2D/CD8a/2B4/CD3zeta chimeric receptor, which comprises an NKG2Dfragment extracellular receptor domain that binds native ligands ofNKG2D, a CD8a transmembrane region, and an effector domain comprisingthe signaling domains of CD3zeta and 2B4. Additionally, in severalembodiments, this construct can optionally be co-expressed with mbIL15.As discussed above, 4-1BB, like CD3zeta and 2B4, can function as apotent activator of immune signaling cascades. In several embodiments,these three signaling components act in a synergistic manner to activateNK cells upon binding of a ligand to the chimeric receptor. Thus, inseveral embodiments, there are provided polynucleotides encoding aNKG2D/CD8a/4-1BB/2B4/CD3zeta chimeric receptor, which comprises an NKG2Dfragment extracellular receptor domain that binds native ligands ofNKG2D, a CD8 transmembrane region, and an effector domain comprising thesignaling domains of 4-1BB, 2B4 and CD3zeta. In one embodiment, thischimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO:58. In yet another embodiment, the NKG2D-CD8a-4-1BB-CD3zeta chimericreceptor comprises the amino acid sequence of SEQ ID NO: 59. In severalembodiments, this construct is particularly efficacious when the NKcells concurrently express mbIL15, the mbIL15 provides a furthersynergistic effect with respect to the activation and/or cytotoxicnature of the NK cells. In some embodiments, the sequence of thechimeric receptor may vary from SEQ ID NO. 58, but remains, depending onthe embodiment, at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, or at least 95% homologous with SEQ ID NO. 58. In severalembodiments, while the chimeric receptor may vary from SEQ ID NO. 58,the chimeric receptor retains, or in some embodiments, has enhanced, NKcell activating and/or cytotoxic function.

In several alternative embodiments, there are provided polynucleotidesencoding a NKG2D/CD8a/DAP10/4-1BB chimeric receptor, which comprises anNKG2D fragment extracellular receptor domain that binds native ligandsof NKG2D, a CD8a transmembrane region, and an effector domain comprisingthe signaling domains of 4-1BB and DAP10. In one embodiment, thischimeric receptor is encoded by the nucleic acid sequence of SEQ ID NO:60. In yet another embodiment, the NKG2D-CD8a-4-1BB-DAP10 chimericreceptor comprises the amino acid sequence of SEQ ID NO: 61.Additionally, in several embodiments, this construct can optionally beco-expressed with mbIL15. In several embodiments, this construct isparticularly efficacious when the NK cells concurrently express mbIL15,the mbIL15 provides a further synergistic effect with respect to theactivation and cytotoxic nature of the NK cells. In some embodiments,the sequence of the chimeric receptor may vary from SEQ ID NO. 60, butremains, depending on the embodiment, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% homologous withSEQ ID NO. 60. In several embodiments, while the chimeric receptor mayvary from SEQ ID NO. 60, the chimeric receptor retains, or in someembodiments, has enhanced, NK cell activating and/or cytotoxic function.Further, as discussed above, 2B4, like DAP10 and 4-1BB, is a potentactivator of immune signaling cascades. In several embodiments, thesethree signaling components act in a synergistic manner to activate NKcells upon binding of a ligand to the chimeric receptor. Thus, inseveral embodiments, there are provided polynucleotides encoding aNKG2D/CD8a/4-1BB/DAP10/2B4 chimeric receptor, which comprises an NKG2Dfragment extracellular receptor domain that binds native ligands ofNKG2D, a CD8 transmembrane region, and an effector domain comprising thesignaling domains of 4-1BB, 2B4 and DAP10, wherein 4-1BB is followed byDAP10, and DAP10 is followed by 2B4. In one embodiment, this chimericreceptor is encoded by the nucleic acid sequence of SEQ ID NO: 62. Inyet another embodiment, the NKG2D-CD8a-4-1BB-CD3zeta chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 63. In severalembodiments, this construct is particularly efficacious when the NKcells concurrently express mbIL15, the mbIL15 provides a furthersynergistic effect with respect to the activation and cytotoxic natureof the NK cells. In some embodiments, the sequence of the chimericreceptor may vary from SEQ ID NO. 62, but remains, depending on theembodiment, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% homologous with SEQ ID NO. 62. In severalembodiments, while the chimeric receptor may vary from SEQ ID NO. 62,the chimeric receptor retains, or in some embodiments, has enhanced, NKcell activating and/or cytotoxic function. In several other embodiments,there are provided polynucleotides encoding a NKG2D/CD8a/4-1BB/2B4/DAP10chimeric receptor, which comprises an NKG2D fragment extracellularreceptor domain that binds native ligands of NKG2D, a CD8 transmembraneregion, and an effector domain comprising the signaling domains of4-1BB, 2B4 and DAP10, wherein 4-1BB is followed by 2B4, and 2B4 isfollowed by DAP10. In one embodiment, this chimeric receptor is encodedby the nucleic acid sequence of SEQ ID NO: 64. In yet anotherembodiment, the NKG2D-CD8a-4-1BB-CD3zeta chimeric receptor comprises theamino acid sequence of SEQ ID NO: 65. In several embodiments, thisconstruct is particularly efficacious when the NK cells concurrentlyexpress mbIL15, the mbIL15 provides a further synergistic effect withrespect to the activation and cytotoxic nature of the NK cells. In someembodiments, the sequence of the chimeric receptor may vary from SEQ IDNO. 64, but remains, depending on the embodiment, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 64. In several embodiments, while thechimeric receptor may vary from SEQ ID NO. 64, the chimeric receptorretains, or in some embodiments, has enhanced, NK cell activating and/orcytotoxic function.

In several additional embodiments, transmembrane and effector domains(and associated function) of the chimeric receptor are derived from thesame peptide. CD16 is a potent activating receptor expressed on thesurface of NK cells. Thus, in several embodiments, polynucleotides areprovided encoding a NKG2D/CD16 chimeric receptor, which comprises anNKG2D fragment extracellular receptor domain that binds native ligandsof NKG2D and a CD16 peptide comprising both the transmembrane region andintracellular effector domain. In one embodiment, this chimeric receptorcomprises the nucleic acid sequence of SEQ ID NO: 23. In yet anotherembodiment, this chimeric receptor is encoded by the amino acid sequenceof SEQ ID NO: 24. In some embodiments, the sequence of the chimericreceptor may vary from SEQ ID NO. 23, but remains, depending on theembodiment, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% homologous with SEQ ID NO. 23. In severalembodiments, while the chimeric receptor may vary from SEQ ID NO. 23,the chimeric receptor retains, or in some embodiments, has enhanced, NKcell activating and/or cytotoxic function. Additionally, in severalembodiments, this construct can optionally be co-expressed with mbIL15.

In several additional embodiments, polynucleotides are provided encodinga NKG2D/CD16/4-1BB chimeric receptor, wherein the signaling domain of4-1BB acts as a second transducer of activating signals in the effectordomain. Additionally, in several embodiments, this construct canoptionally be co-expressed with mbIL15.

CD3zeta dimerizes via its transmembrane domain. Thus, in severalembodiments, chimeric receptors are provided wherein a CD3zetatransmembrane domain recruits full-length CD3zeta molecule to thesynapse. In several embodiments, there are provided polynucleotidesencoding a chimeric receptor which comprises a NKG2D fragment that bindsnative ligands of NKG2D, a CD8a hinge, 0, 1, 2, 3, 4, 5, 6 or moreextracellular CD3zeta residues (the “juxta-membrane portion”) directlyadjacent to a CD3zeta transmembrane domain, and an effector domaincomprising one or more of CD16, NCR1, NCR2, NCR3, 4-1BB, NKp80, FcRγ,CD3zeta and 2B4.

In several embodiments, chimeric receptors are provided wherein aCD3zeta transmembrane domain is coupled to an effector domain comprisingone or both of 4-1BB and CD16. Thus, in several embodiments,polynucleotides are provided encoding a NKG2D/CD3zetaTM/4-1BB chimericreceptor, which comprises a fragment of NKG2D that is codon optimizedcoupled to a CD8a hinge, a CD3zeta transmembrane region, and an effectordomain comprising 4-1BB. In one embodiment, this chimeric receptorcomprises the nucleic acid sequence of SEQ ID NO: 78. In yet anotherembodiment, this chimeric receptor is encoded by the amino acid sequenceof SEQ ID NO: 79. In some embodiments, the sequence of the chimericreceptor may vary from SEQ ID NO. 78, but remains, depending on theembodiment, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% homologous with SEQ ID NO. 78. In severalembodiments, while the chimeric receptor may vary from SEQ ID NO. 78,the chimeric receptor retains, or in some embodiments, has enhanced, NKcell activating and/or cytotoxic function. Additionally, in severalembodiments, this construct can optionally be co-expressed with mbIL15.

In several embodiments, polynucleotides are provided encoding aNKG2D/CD3zetaTM/CD16/4-1BB chimeric receptor, which comprises a fragmentof NKG2D that is codon optimized coupled to a CD8a hinge, a CD3zetatransmembrane region, and an effector domain comprising CD16 followed by4-1BB. In one embodiment, this chimeric receptor comprises the nucleicacid sequence of SEQ ID NO: 70. In yet another embodiment, this chimericreceptor is encoded by the amino acid sequence of SEQ ID NO: 71. In someembodiments, the sequence of the chimeric receptor may vary from SEQ IDNO. 70, but remains, depending on the embodiment, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 70. In several embodiments, while thechimeric receptor may vary from SEQ ID NO. 70, the chimeric receptorretains, or in some embodiments, has enhanced, NK cell activating and/orcytotoxic function. Additionally, in several embodiments, this constructcan optionally be co-expressed with mbIL15. Further, in severalembodiments, polynucleotides are provided encoding aNKG2D/CD3zetaTM/4-1BB/CD16 chimeric receptor, which comprises a fragmentof NKG2D that is codon optimized coupled to a CD8a hinge, a CD3zetatransmembrane region, and an effector domain comprising 4-1BB followedby CD16. In some embodiments, the effector domain further comprises aGS3 linker. In some embodiments, the GS3 linker is positioned between4-1BB and CD16. In one embodiment, this chimeric receptor comprises thenucleic acid sequence of SEQ ID NO: 84. In yet another embodiment, thischimeric receptor is encoded by the amino acid sequence of SEQ ID NO:85. In some embodiments, the sequence of the chimeric receptor may varyfrom SEQ ID NO. 84, but remains, depending on the embodiment, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% homologous with SEQ ID NO. 84. In several embodiments, while thechimeric receptor may vary from SEQ ID NO. 84, the chimeric receptorretains, or in some embodiments, has enhanced, NK cell activating and/orcytotoxic function. Additionally, in several embodiments, this constructcan optionally be co-expressed with mbIL15. Further, in severalembodiments, polynucleotides are provided encoding aNKG2Dx2/CD3zetaTM/CD16/4-1BB chimeric receptor, which comprises thefragment of NKG2D that is codon optimized coupled to a GS3 linker, anadditional NKG2D fragment, a CD8a hinge, a CD3zeta transmembrane region,and an effector domain comprising a CD16 and 4-1BB. In one embodiment,this chimeric receptor comprises the nucleic acid sequence of SEQ ID NO:72. In yet another embodiment, this chimeric receptor is encoded by theamino acid sequence of SEQ ID NO: 73. In some embodiments, the sequenceof the chimeric receptor may vary from SEQ ID NO. 72, but remains,depending on the embodiment, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, or at least 95% homologous with SEQ ID NO.72. In several embodiments, while the chimeric receptor may vary fromSEQ ID NO. 72, the chimeric receptor retains, or in some embodiments,has enhanced, NK cell activating and/or cytotoxic function.Additionally, in several embodiments, this construct can optionally beco-expressed with mbIL15.

In several embodiments, chimeric receptors are provided wherein aCD3zeta transmembrane domain is coupled to an effector domain comprisingNKp80. Thus, in several embodiments, polynucleotides are providedencoding a NKG2D/CD3zetaTM/CD16/4-1BB/NKp80 chimeric receptor, whichchimeric receptor comprises a fragment of NKG2D coupled to a CD8a hinge,a CD3zeta transmembrane region, and an effector domain comprising aCD16, 4-1BB, and NKp80. In some embodiments, the effector domain furthercomprises a GS3 linker. In some embodiments, the GS3 linker ispositioned between 4-1BB and NKp80. In one embodiment, this chimericreceptor comprises the nucleic acid sequence of SEQ ID NO: 74. In yetanother embodiment, this chimeric receptor is encoded by the amino acidsequence of SEQ ID NO: 75. In some embodiments, the sequence of thechimeric receptor may vary from SEQ ID NO. 74, but remains, depending onthe embodiment, at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, or at least 95% homologous with SEQ ID NO. 74. In severalembodiments, while the chimeric receptor may vary from SEQ ID NO. 74,the chimeric receptor retains, or in some embodiments, has enhanced, NKcell activating and/or cytotoxic function. Additionally, in severalembodiments, this construct can optionally be co-expressed with mbIL15.Further, in several embodiments, polynucleotides are provided encoding a2xNKG2D/CD3zetaTM/CD16/4-1BB/NKp80 chimeric receptor, which comprisesthe fragment of NKG2D that is codon optimized coupled to a GS3 linker,an additional NKG2D fragment, a CD8a hinge, a CD3zeta transmembraneregion, and an effector domain comprising a CD16, 4-1BB, and NKp80. Insome embodiments, the effector domain further comprises a GS3 linker. Insome embodiments, the GS3 linker is positioned between 4-1BB and NKp80.In one embodiment, this chimeric receptor comprises the nucleic acidsequence of SEQ ID NO: 76. In yet another embodiment, this chimericreceptor is encoded by the amino acid sequence of SEQ ID NO: 77. In someembodiments, the sequence of the chimeric receptor may vary from SEQ IDNO. 76, but remains, depending on the embodiment, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 76. In several embodiments, while thechimeric receptor may vary from SEQ ID NO. 76, the chimeric receptorretains, or in some embodiments, has enhanced, NK cell activating and/orcytotoxic function. Additionally, in several embodiments, this constructcan optionally be co-expressed with mbIL15. Further, in severalembodiments, polynucleotides are provided encoding aNKG2D/CD3zetaTM/4-1BB/NKp80 chimeric receptor, which comprises afragment of NKG2D that is codon optimized coupled to a CD8a hinge, aCD3zeta transmembrane region, and an effector domain comprising 4-1BBand NKp80. In some embodiments, the effector domain further comprises aGS3 linker. In some embodiments, the GS3 linker is positioned between4-1BB and NKp80. In one embodiment, this chimeric receptor comprises thenucleic acid sequence of SEQ ID NO: 82. In yet another embodiment, thischimeric receptor is encoded by the amino acid sequence of SEQ ID NO:83. In some embodiments, the sequence of the chimeric receptor may varyfrom SEQ ID NO. 82, but remains, depending on the embodiment, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% homologous with SEQ ID NO. 82. In several embodiments, while thechimeric receptor may vary from SEQ ID NO. 82, the chimeric receptorretains, or in some embodiments, has enhanced, NK cell activating and/orcytotoxic function. Additionally, in several embodiments, this constructcan optionally be co-expressed with mbIL15.

In several embodiments, chimeric receptors are provided wherein aCD3zeta transmembrane domain is coupled to an effector domain comprisingCD3zeta. Thus, in several embodiments, polynucleotides are providedencoding a NKG2D/CD3zetaTM/4-1BB/CD3zeta chimeric receptor, whichcomprises a fragment of NKG2D that is codon optimized coupled to a CD8ahinge, a CD3zeta transmembrane region, and an effector domain comprising4-1BB and CD3zeta. In one embodiment, this chimeric receptor comprisesthe nucleic acid sequence of SEQ ID NO: 80. In yet another embodiment,this chimeric receptor is encoded by the amino acid sequence of SEQ IDNO: 81. In some embodiments, the sequence of the chimeric receptor mayvary from SEQ ID NO. 80, but remains, depending on the embodiment, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95% homologous with SEQ ID NO. 80. In several embodiments, whilethe chimeric receptor may vary from SEQ ID NO. 80, the chimeric receptorretains, or in some embodiments, has enhanced, NK cell activating and/orcytotoxic function. Additionally, in several embodiments, this constructcan optionally be co-expressed with mbIL15.

In several embodiments, chimeric receptors are provided wherein aCD3zeta transmembrane domain is coupled to an effector domain comprisingFcRγ. Thus, in several embodiments, polynucleotides are providedencoding a NKG2D/CD3zetaTM/4-1BB/FcRγ chimeric receptor, which comprisesa fragment of NKG2D coupled to a CD8a hinge, a CD3zeta transmembraneregion, and an effector domain comprising 4-1BB and FcRγ. In oneembodiment, this chimeric receptor comprises the nucleic acid sequenceof SEQ ID NO: 86. In yet another embodiment, this chimeric receptor isencoded by the amino acid sequence of SEQ ID NO: 87. In someembodiments, the sequence of the chimeric receptor may vary from SEQ IDNO. 86, but remains, depending on the embodiment, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 86. In several embodiments, while thechimeric receptor may vary from SEQ ID NO. 86, the chimeric receptorretains, or in some embodiments, has enhanced, NK cell activating and/orcytotoxic function. Additionally, in several embodiments, this constructcan optionally be co-expressed with mbIL15.

In several embodiments, chimeric receptors are provided wherein aCD3zeta transmembrane domain is coupled to an effector domain comprisingCD28. Thus, in several embodiments, polynucleotides are providedencoding a NKG2D/CD3zetaTM/CD28/CD3zeta chimeric receptor, whichcomprises an NKG2D fragment extracellular receptor domain that bindsnative ligands of NKG2D, a CD8a hinge, a CD3zeta transmembrane region,and intracellular effector domain comprising CD28 and CD3zeta. In oneembodiment, this chimeric receptor comprises the nucleic acid sequenceof SEQ ID NO: 102. In yet another embodiment, this chimeric receptor isencoded by the amino acid sequence of SEQ ID NO: 103. In someembodiments, the sequence of the chimeric receptor may vary from SEQ IDNO. 102, but remains, depending on the embodiment, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 102. In several embodiments, while thechimeric receptor may vary from SEQ ID NO. 102, the chimeric receptorretains, or in some embodiments, has enhanced, NK cell activating and/orcytotoxic function. Additionally, in several embodiments, this constructcan optionally be co-expressed with mbIL15.

In several embodiments, chimeric receptors are provided wherein theextracellular domain comprises a fragment of NKG2D coupled IL15. Thus,in several embodiments, polynucleotides are provided encoding anIL15/NKG2D/CD8a/4-1BB/CD3zeta chimeric receptor, which comprises anNKG2D fragment extracellular receptor domain that binds native ligandsof NKG2D linked to IL-15, a CD8a hinge, a CD8a transmembrane domain, andintracellular effector domain comprising 4-1BB and CD3z. In someembodiments, the extracellular domain further comprises a GS3 linker. Insome embodiments, the GS3 linker is positioned between IL15 and theNKG2D fragment extracellular receptor domain. In one embodiment, thischimeric receptor comprises the nucleic acid sequence of SEQ ID NO: 88.In yet another embodiment, this chimeric receptor is encoded by theamino acid sequence of SEQ ID NO: 89. In some embodiments, the sequenceof the chimeric receptor may vary from SEQ ID NO. 88, but remains,depending on the embodiment, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, or at least 95% homologous with SEQ ID NO.88. In several embodiments, while the chimeric receptor may vary fromSEQ ID NO. 88, the chimeric receptor retains, or in some embodiments,has enhanced, NK cell activating and/or cytotoxic function.

In several embodiments, chimeric receptors are provided wherein theextracellular domain comprises a fragment of NKG2D coupled to a IgG4short hinge. Thus, in several embodiments, polynucleotides are providedencoding a NKG2D/IgG4/CD8a/4-1BB/CD3zeta chimeric receptor, whichcomprises an NKG2D fragment extracellular receptor domain that bindsnative ligands of NKG2D, an IgG4 short hinge, a CD8a transmembranedomain, and intracellular effector domain comprising 4-1BB, and CD3zeta.In one embodiment, this chimeric receptor comprises the nucleic acidsequence of SEQ ID NO: 96. In yet another embodiment, this chimericreceptor is encoded by the amino acid sequence of SEQ ID NO: 97. In someembodiments, the sequence of the chimeric receptor may vary from SEQ IDNO. 96, but remains, depending on the embodiment, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 96. In several embodiments, while thechimeric receptor may vary from SEQ ID NO. 96, the chimeric receptorretains, or in some embodiments, has enhanced, NK cell activating and/orcytotoxic function. Additionally, in several embodiments, this constructcan optionally be co-expressed with mbIL15.

In several embodiments, chimeric receptors are provided wherein theeffector domain comprises 0X40. Thus, in several embodiments,polynucleotides are provided encoding a NKG2D/CD8a/OX40/CD3z chimericreceptor, which comprises an NKG2D fragment extracellular receptordomain that binds native ligands of NKG2D, a CD8a hinge, a CD8atransmembrane domain, and an intracellular effector domain comprisingOX40, and CD3z. In one embodiment, this chimeric receptor comprises thenucleic acid sequence of SEQ ID NO: 90. In yet another embodiment, thischimeric receptor is encoded by the amino acid sequence of SEQ ID NO:91. In some embodiments, the sequence of the chimeric receptor may varyfrom SEQ ID NO. 90, but remains, depending on the embodiment, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% homologous with SEQ ID NO. 90. In several embodiments, while thechimeric receptor may vary from SEQ ID NO. 90, the chimeric receptorretains, or in some embodiments, has enhanced, NK cell activating and/orcytotoxic function. Additionally, in several embodiments, this constructcan optionally be co-expressed with mbIL15. In several embodiments,polynucleotides are provided encoding a NKG2D/IgG4/CD8a/OX40/CD3zetachimeric receptor, which comprises an NKG2D fragment extracellularreceptor domain that binds native ligands of NKG2D, an IgG4 hinge, aCD8a transmembrane domain, and intracellular effector domain comprisingOX40 and CD3zeta. In one embodiment, this chimeric receptor comprisesthe nucleic acid sequence of SEQ ID NO: 100. In yet another embodiment,this chimeric receptor is encoded by the amino acid sequence of SEQ IDNO: 101. In some embodiments, the sequence of the chimeric receptor mayvary from SEQ ID NO. 100, but remains, depending on the embodiment, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95% homologous with SEQ ID NO. 100. In several embodiments, whilethe chimeric receptor may vary from SEQ ID NO. 100, the chimericreceptor retains, or in some embodiments, has enhanced, NK cellactivating and/or cytotoxic function. Additionally, in severalembodiments, this construct can optionally be co-expressed with mbIL15.

In several embodiments, chimeric receptors are provided comprising aCD28 peptide comprising both the transmembrane region and intracellulareffector domain. Thus, in several embodiments, polynucleotides areprovided encoding a NKG2D/CD28/CD3zeta chimeric receptor, whichcomprises an NKG2D fragment extracellular receptor domain that bindsnative ligands of NKG2D, a CD8a hinge, a CD28transmembrane/intracellular domain, and CD3zeta. In one embodiment, thischimeric receptor comprises the nucleic acid sequence of SEQ ID NO: 92.In yet another embodiment, this chimeric receptor is encoded by theamino acid sequence of SEQ ID NO: 93. In some embodiments, the sequenceof the chimeric receptor may vary from SEQ ID NO. 92, but remains,depending on the embodiment, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, or at least 95% homologous with SEQ ID NO.92. In several embodiments, while the chimeric receptor may vary fromSEQ ID NO. 92, the chimeric receptor retains, or in some embodiments,has enhanced, NK cell activating and/or cytotoxic function.Additionally, in several embodiments, this construct can optionally beco-expressed with mbIL15. In further embodiments, polynucleotides areprovided encoding a NKG2D/CD28/CD3zeta/4-1BB chimeric receptor, whichcomprises an NKG2D fragment extracellular receptor domain that bindsnative ligands of NKG2D, a CD8a hinge, a CD28transmembrane/intracellular domain, and 4-1BB and CD3zeta. In oneembodiment, this chimeric receptor comprises the nucleic acid sequenceof SEQ ID NO: 94. In yet another embodiment, this chimeric receptor isencoded by the amino acid sequence of SEQ ID NO: 95. In someembodiments, the sequence of the chimeric receptor may vary from SEQ IDNO. 94, but remains, depending on the embodiment, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 94. In several embodiments, while thechimeric receptor may vary from SEQ ID NO. 94, the chimeric receptorretains, or in some embodiments, has enhanced, NK cell activating and/orcytotoxic function. Additionally, in several embodiments, this constructcan optionally be co-expressed with mbIL15. In further embodiments,polynucleotides are provided encoding a NKG2D/IgG4/CD28/CD3zeta chimericreceptor, which comprises an NKG2D fragment extracellular receptordomain that binds native ligands of NKG2D, an IgG4 hinge, a CD28transmembrane/intracellular domain, and CD3zeta. In one embodiment, thischimeric receptor comprises the nucleic acid sequence of SEQ ID NO: 98.In yet another embodiment, this chimeric receptor is encoded by theamino acid sequence of SEQ ID NO: 99. In some embodiments, the sequenceof the chimeric receptor may vary from SEQ ID NO. 98, but remains,depending on the embodiment, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, or at least 95% homologous with SEQ ID NO.98. In several embodiments, while the chimeric receptor may vary fromSEQ ID NO. 98, the chimeric receptor retains, or in some embodiments,has enhanced, NK cell activating and/or cytotoxic function.Additionally, in several embodiments, this construct can optionally beco-expressed with mbIL15.

NCR1 (NKp46), NCR2 (NKp44) and NCR3 (NKp30) are receptors on NK cellsthat transduce activation signals upon ligand binding. Thus, in severalembodiments, polynucleotides are provided encoding a NKG2D/NCR1 chimericreceptor, which comprises an NKG2D fragment extracellular receptordomain that binds native ligands of NKG2D and a NCR1 peptide comprisingboth the transmembrane region and intracellular effector domain. In oneembodiment, this chimeric receptor comprises the nucleic acid sequenceof SEQ ID NO: 27. In yet another embodiment, this chimeric receptor isencoded by the amino acid sequence of SEQ ID NO: 28. In someembodiments, the sequence of the chimeric receptor may vary from SEQ IDNO. 30, but remains, depending on the embodiment, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 27. In several embodiments, while thechimeric receptor may vary from SEQ ID NO. 27, the chimeric receptorretains, or in some embodiments, has enhanced, NK cell activating and/orcytotoxic function. Additionally, in several embodiments, this constructcan optionally be co-expressed with mbIL15.

In several additional embodiments, polynucleotides are provided encodinga NKG2D/NCR1/4-1BB chimeric receptor, wherein the signaling domain of4-1BB acts as a second transducer of activating signals in the effectordomain, leading to synergistically enhanced NK cell activation andcytotoxicity. In several additional embodiments, polynucleotides areprovided encoding a NKG2D/NCR2 chimeric receptor, which comprises anNKG2D fragment extracellular receptor domain that binds native ligandsof NKG2D and a NCR2 peptide comprising both the transmembrane region andintracellular effector domain. As with NCR1, in several embodimentsthese constructs are particularly amenable for use in creating NK cellsexpressing the chimeric receptor, due to their relatively small size andsimplicity on sequence. However, they retain the ability, in severalembodiments, to yield highly effective NK cells, despite the apparentsimplicity of the construct. Additionally, in several embodiments, theseconstructs can optionally be co-expressed with mbIL15.

In several additional embodiments, polynucleotides are provided encodinga NKG2D/NCR3 chimeric receptor, which comprises an NKG2D fragmentextracellular receptor domain that binds native ligands of NKG2D and aNCR3 peptide comprising both the transmembrane region and intracellulareffector domain. As with NCR1 and or NCR2, in several embodiments theseconstructs are particularly amenable for use in creating NK cellsexpressing the chimeric receptor, due to their relatively small size andsimplicity on sequence. However, they retain the ability, in severalembodiments, to yield highly effective NK cells, despite the apparentsimplicity of the construct. In one embodiment, this chimeric receptorcomprises the nucleic acid sequence of SEQ ID NO: 29. In yet anotherembodiment, this chimeric receptor is encoded by the amino acid sequenceof SEQ ID NO: 30. In some embodiments, the sequence of the chimericreceptor may vary from SEQ ID NO. 29, but remains, depending on theembodiment, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% homologous with SEQ ID NO. 29. In severalembodiments, while the chimeric receptor may vary from SEQ ID NO. 29,the chimeric receptor retains, or in some embodiments, has enhanced, NKcell activating and/or cytotoxic function. Additionally, in severalembodiments, this construct can optionally be co-expressed with mbIL15.

In several additional embodiments, polynucleotides are provided encodinga NKG2D/NCR2/4-1BB chimeric receptor, wherein the signaling domain of4-1BB acts as a second transducer of activating signals in the effectordomain, thereby leading to a synergistic effect between the signalingdomains, and unexpectedly effectively cytotoxic NK cells. Additionally,in several embodiments, this construct can optionally be co-expressedwith mbIL15.

In several additional embodiments, polynucleotides are provided encodinga NKG2D/NCR3/4-1BB chimeric receptor, wherein the signaling domain of4-1BB acts as a second transducer of activating signals in the effectordomain, thereby leading to a synergistic effect between the signalingdomains, and unexpectedly effectively cytotoxic NK cells. Additionally,in several embodiments, this construct can optionally be co-expressedwith mbIL15.

In some embodiments the surface expression and efficacy of the chimericreceptors disclosed herein are enhanced by variations in a spacer region(hinge), which, in several embodiments, are located in the extracellulardomain between the NKG2D fragment and the transmembrane domain. In someembodiments, the hinge regions can be included between other portions ofthe chimeric receptor (e.g., between intracellular and transmembranedomains, or between multiple intracellular domains). In someembodiments, domains that serve certain purposes as disclosed elsewhereherein, can serve additional functions. For example, in severalembodiments, CD8a is repurposed to serve as a hinge region (encoded, inseveral embodiments, by the nucleic acid sequence of SEQ ID NO: 5). Inyet another embodiment, the hinge region comprises an N-terminaltruncated form of CD8a and/or a C-terminal truncated form of CD8a.Depending on the embodiment, these truncations can be at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90% homologous to the hinge encoded by SEQ ID NO. 5. Inseveral additional embodiments, the hinge comprises spans of Glycine andSerine residues (herein termed “GS linkers”) where GSn represents thesequence (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO. 42). In one embodiment, thehinge comprises both CD8a and GS3, and is encoded by the amino acidsequence of SEQ ID NO: 32, for example, where n=3. In additionalembodiments, the value of n may be equal to 1, 2, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15 or greater depending on the embodiment. In severalembodiments, the hinge could also be structured as GSn/CD8a.Alternatively, the GS linker can comprise the entire hinge region. Inone such embodiment, the hinge region is encoded by the nucleic acidsequence of SEQ ID NO: 33. In another such embodiment, the hinge regionis encoded by the nucleic acid sequence of SEQ ID NO: 34. In severalembodiments, IgG4 is repurposed as a hinge region (encoded, in severalembodiments, by the nucleic acid sequence of SEQ ID NO: 104). In yetanother embodiment, the hinge region comprises an N-terminal truncatedform of IgG4 and/or a C-terminal truncated form of IgG4. Depending onthe embodiment, these truncations can be at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, or at least about 90%homologous to the hinge encoded by SEQ ID NO. 104.

In several embodiments, the chimeric receptor constructs employ a 2B4intracellular signaling domain. In several embodiments, this domain isencoded by the amino acid sequence of SEQ ID NO. 35. In someembodiments, the 2B4 domain is encoded by the nucleic acid sequence ofSEQ ID NO. 36. In some embodiments, the sequence of the 2B4intracellular domain used in a chimeric receptor may vary from SEQ IDNO. 36, but remains, depending on the embodiment, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 36. In several embodiments, while thesignaling domain of the chimeric receptor may vary from SEQ ID NO. 36,the chimeric receptor retains, or in some embodiments, has enhanced, NKcell activating and/or cytotoxic function. Likewise, in severalembodiments an NKp80 intracellular domain is used, in severalembodiments. In some embodiments, the NKp80 domain is the soleintracellular signaling domain, while in some embodiments, that domainis used in conjunction with one or more additional domains. In severalembodiments, the NKp80 is encoded by the amino acid sequence of SEQ IDNO. 37. In some embodiments, the NKp80 domain is encoded by the nucleicacid sequence of SEQ ID NO. 38. In some embodiments, the sequence of theNKp80 intracellular domain used in a chimeric receptor may vary from SEQID NO. 38, but remains, depending on the embodiment, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 38. In several embodiments, while thesignaling domain of the chimeric receptor may vary from SEQ ID NO. 38,the chimeric receptor retains, or in some embodiments, has enhanced, NKcell activating and/or cytotoxic function.

In several embodiments, the chimeric receptor uses a portion of abeta-adrenergic receptor as a transmembrane domain. In severalembodiments, the portion comprises a portion of the beta-adrenergicextracellular domain. In several embodiments, the portion is a portionof the beta-adrenergic receptor transmembrane domain. In severalembodiments, a combination of an extracellular domain and atransmembrane domain of the beta adrenergic receptor is used. Dependingon the embodiment the portions are from the beta-1 and/or beta-2adrenergic receptor. In several embodiments, a portion of the N-terminalextracellular region of the beta-2 adrenergic receptor is used. Inseveral embodiments that portion has the amino acid sequence of SEQ IDNO. 39. In some embodiments, the extracellular beta-2 adrenergic domainis encoded by the nucleic acid sequence of SEQ ID NO. 40. In someembodiments, the sequence of the extracellular beta-2 adrenergic domainused in a chimeric receptor may vary from SEQ ID NO. 39, but remains,depending on the embodiment, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, or at least 95% homologous with SEQ ID NO.39. In several embodiments, the first transmembrane helix of the beta-2adrenergic receptor is used, optionally in conjunction with theextracellular beta-2 adrenergic domain. In several embodiments, thefirst transmembrane helix of the beta-2 adrenergic receptor has theamino acid sequence of SEQ ID NO. 41. In some embodiments, the firsttransmembrane helix of the beta-2 adrenergic receptor is encoded by thenucleic acid sequence of SEQ ID NO. 42. In some embodiments, thesequence of the first transmembrane helix of the beta-2 adrenergicreceptor used in a chimeric receptor may vary from SEQ ID NO. 41, butremains, depending on the embodiment, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% homologous withSEQ ID NO. 41.

In one embodiment, the chimeric receptor comprises CD8, truncated NKG2D,CD8a, transmembrane domain, a CD16 intracellular domain, and 4-1BB as acostimulatory molecule. In several embodiments, such a construct isencoded by SEQ ID NO. 25. In some embodiments, the chimeric receptor mayvary from SEQ ID NO. 25, but remains, depending on the embodiment, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95% homologous with SEQ ID NO. 25. In several embodiments, hingeregions surrounding CD8 are increased by way of addition of GS linkers(disclosed herein), such as GS3, by way of non-limiting example. In suchembodiments, the construct is encoded by the nucleic acid of SEQ ID NO.43. In some embodiments, the chimeric receptor may vary from SEQ ID NO.43, but remains, depending on the embodiment, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 43. In several embodiments, hinge regionssurrounding CD8 are increased by way of addition of longer GS linkers,such as GS12, or other linker. In several embodiments, hinge regions aredecreased by way of truncating CD8. For example, in several embodiments,the N-terminal region of CD8a is truncated by at least 20%, at least30%, at least 40%, or at least 50%. In several embodiments, the CD8hinge is replaced with a GS linker. For example, in several embodiments,the hinge region comprises a GS3 linker, thereby the construct comprisesNKG2D-GS3-CD16-4-1BB. In one embodiment, such a construct is encoded bythe nucleic acid of SEQ ID NO. 44. In some embodiments, the chimericreceptor may vary from SEQ ID NO. 44, but remains, depending on theembodiment, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% homologous with SEQ ID NO. 44. In severalembodiments, neither CD8 nor GSn are used. In one embodiment, such aconstruct is encoded by the nucleic acid of SEQ ID NO. 45. In someembodiments, the chimeric receptor may vary from SEQ ID NO. 45, butremains, depending on the embodiment, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% homologous withSEQ ID NO. 45.

As discussed above, in several embodiments, codon optimized sequencesare employed. For example in several embodiments, codon optimization(full or partial) is performed on the NKG2D domain of a chimericreceptor. In several embodiments, however, codon optimization is notperformed. In several embodiments, a chimeric receptor construct isprovided with an NKG2D extracellular domain that is not optimized, aCD8a hinge, and a 4-1BB signaling domain. In several embodiments, achimeric receptor construct is provided with an NKG2D extracellulardomain that is not optimized, a CD8a hinge and transmembrane domain, anda 4-1BB signaling domain. In several embodiments, a chimeric receptorconstruct is provided with an NKG2D extracellular domain that is notoptimized, a CD8a hinge and transmembrane domain, a 4-1BB signalingdomain and a 2B4 signaling domain. In several embodiments, such aconstruct has the nucleic acid sequence of SEQ ID NO. 46. In someembodiments, the chimeric receptor may vary from SEQ ID NO. 46, butremains, depending on the embodiment, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% homologous withSEQ ID NO. 46.

In several embodiments, a chimeric receptor construct is provided withan NKG2D extracellular domain that is not optimized, a beta-adrenergicderived transmembrane domain, and a 4-1BB signaling domain. In severalembodiments, a chimeric receptor construct is provided with an NKG2Dextracellular domain that is not optimized, a beta-adrenergic derivedtransmembrane domain made up of the extracellular region of the beta-2adrenergic receptor and the first transmembrane helix of the beta-2adrenergic receptor, and a 4-1BB signaling domain. In severalembodiments, a chimeric receptor construct is provided with an NKG2Dextracellular domain that is not optimized, a beta-adrenergic derivedtransmembrane domain made up of the extracellular region of the beta-2adrenergic receptor and the first transmembrane helix of the beta-2adrenergic receptor, a 4-1BB signaling domain and a 2B4 signalingdomain. In several embodiments, such a construct has the nucleic acidsequence of SEQ ID NO. 47. In some embodiments, the chimeric receptormay vary from SEQ ID NO. 47, but remains, depending on the embodiment,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, orat least 95% homologous with SEQ ID NO. 47.

In several embodiments, a chimeric receptor construct is provided withan NKG2D extracellular domain that is not optimized, a CD8a hinge, and a2B4 signaling domain. In several embodiments, a chimeric receptorconstruct is provided with an NKG2D extracellular domain that is notoptimized, a CD8a hinge and transmembrane domain, and both a 2B4 and a4-1BB signaling domain. In several embodiments, a chimeric receptorconstruct is provided with an NKG2D extracellular domain that is notoptimized, a CD8a hinge and transmembrane domain, a 4-1BB signalingdomain and a 2B4 signaling domain, as well as a NKp80 domain. In severalembodiments, a GS linker, such as a GS3 linker joins the 2B4 and NKp80domains. In several embodiments, such a construct has the nucleic acidsequence of SEQ ID NO. 48. In some embodiments, the chimeric receptormay vary from SEQ ID NO. 48, but remains, depending on the embodiment,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, orat least 95% homologous with SEQ ID NO. 48.

In several embodiments, a chimeric receptor construct is provided withan NKG2D extracellular domain that is not optimized, a CD8a hinge, and aNKp80 signaling domain. In several embodiments, a chimeric receptorconstruct is provided with an NKG2D extracellular domain that is notoptimized, a CD8a hinge and transmembrane domain, and a NKp80 signalingdomain. In several embodiments, a chimeric receptor construct isprovided with an NKG2D extracellular domain that is not optimized, aCD8a hinge and transmembrane domain, a 4-1BB signaling domain and aNKp80 domain. In several embodiments, a GS linker, such as a GS3 linkerjoins the 4-1BB and NKp80 domains. In several embodiments, such aconstruct has the nucleic acid sequence of SEQ ID NO. 49. In someembodiments, the chimeric receptor may vary from SEQ ID NO. 49, butremains, depending on the embodiment, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% homologous withSEQ ID NO. 49.

In several embodiments, a CD8 transmembrane domain is coupled with a 2B4intracellular domain. In several embodiments, a CD8 transmembrane domainis replaced with a 2B4 domain that is transmembrane and intracellular.In several embodiments, the CD8 transmembrane domain is replaced with2B4 and 4-1BB is expressed in a proximal configuration.

In several embodiments, a CD16 intracellular signaling domain is coupledwith a CD3zeta or gamma subunit which are exogenously expressed in transto the chimeric receptors described herein. As discussed above, suchconstructs can result in unexpectedly enhanced signal transduction, andthus an unexpected increase in cytotoxic effects of the NK cells.

In several embodiments, the chimeric receptors are configured todimerize, as discussed in additional detail herein. In severalembodiments a truncated NKG2D receptor according to several embodimentsdisclosed herein is optionally dimerized. Dimerization may comprisehomodimers or heterodimers, depending on the embodiment. In severalembodiments, dimerization results in a shift of avidity of the chimericreceptor (and hence the NK cells expressing the receptor) to betterligand recognition with a coordinate balance in reduced (or lack) ofadverse toxic effects. In still further embodiments, the extracellularreceptor domain further comprises a CD8a signal peptide. In severalembodiments, the chimeric receptors employ internal dimers, or repeatsof one or more component subunits. For example, in several embodiments,the chimeric receptor comprises a NKG2D extracellular domain coupled toa second NKG2D extracellular domain, and a transmembrane/signalingregion (or a separate transmembrane region along with a separatesignaling region). In several embodiments, one or more of the NKG2Dextracellular domains are codon optimized. In several embodiments, thetwo NKG2D extracellular domains are separated by a linker, for example aGSn linker. In one embodiment, a GS3 linker is used. In severalembodiments, the transmembrane domain comprises an extracellular regionof the beta-adrenergic receptor. In several embodiments, thetransmembrane domain transmembrane domain comprises an extracellularregion of the beta-2 adrenergic receptor and further comprises the firsttransmembrane domain of the beta-2 adrenergic receptor. In severalembodiments, the signaling region comprises 4-1BB. In severalembodiments, the signaling region comprises NKp80. In severalembodiments, the signaling region comprises a CD16transmembrane-intracellular domain. In several embodiments, thesignaling region comprises 4-1BB in conjunction with NKp80 or a CD16transmembrane-intracellular domain. In several embodiments, the chimericreceptor has the nucleic acid sequence of SEQ ID NO. 50. In someembodiments, the chimeric receptor may vary from SEQ ID NO. 50, butremains, depending on the embodiment, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% homologous withSEQ ID NO. 50. In several embodiments, the chimeric receptor has thenucleic acid sequence of SEQ ID NO. 51. In some embodiments, thechimeric receptor may vary from SEQ ID NO. 51, but remains, depending onthe embodiment, at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, or at least 95% homologous with SEQ ID NO. 51. In severalembodiments, the chimeric receptor has the nucleic acid sequence of SEQID NO. 52. In some embodiments, the chimeric receptor may vary from SEQID NO. 52, but remains, depending on the embodiment, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 52. In several embodiments, the chimericreceptor comprises a hinge region. In several embodiments, CD8a isrepurposed to serve as a hinge region (encoded, in several embodiments,by the nucleic acid sequence of SEQ ID NO: 5). In several embodiments,the chimeric receptor comprises a CD8a transmembrane domain. In severalembodiments, the signaling region comprises 4-1BB in conjunction with2B4 and CD3zeta. In some embodiments, the chimeric receptor comprisesthe fragment of NKG2D that is codon optimized coupled to a GS3 linker,an additional NKG2D fragment, a CD8a hinge, a CD8a transmembrane domain,and an effector domain comprising 4-1BB and CD3zeta. In severalembodiments, the chimeric receptor has the nucleic acid sequence of SEQID NO. 66. In some embodiments, the chimeric receptor may vary from SEQID NO. 66, but remains, depending on the embodiment, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 50. In several embodiments, the chimericreceptor chimeric receptor comprises the amino acid sequence of SEQ IDNO: 67. In some embodiments, the chimeric receptor may vary from SEQ IDNO. 66, but remains, depending on the embodiment, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%homologous with SEQ ID NO. 50.

In several embodiments, the chimeric receptors are configured to bebispecific, as discussed in additional detail herein. In severalembodiments, a truncated NKG2D receptor according to several embodimentsdisclosed herein is bispecific due to a second peptide that binds, forexample, non-NKG2D ligands. In several embodiments, bi-specificityresults in a shift of the targeting of the chimeric receptor (and hencethe NK cells expressing the receptor) to better target cell recognitionwith a coordinate balance in reduced (or lack) of adverse toxic effects.In still further embodiments, the extracellular receptor domain furthercomprises a CD8a signal peptide. For example, in several embodiments,the chimeric receptor comprises a NKG2D extracellular domain coupled toa second extracellular domain that binds other (non-NKG2D) ligands, anda transmembrane/signaling region (or a separate transmembrane regionalong with a separate signaling region). In several embodiments, the twoextracellular domains are separated by a linker, for example a GSnlinker. In one embodiment, a GS3 linker is used.

According to several embodiments disclosed herein, additional chimericreceptors employing codon optimized NKG2D domains are provided for(optionally, these constructs can also be replicated with non-optimizedor partially optimized domains). For example, in several embodiments, acodon optimized extracellular domain is coupled with a hinge and atleast two transmembrane/signaling domains. In several embodiments, themultiple signaling domains provide enhanced cytotoxic efficacy of the NKcells because multiple, non-redundant signal cascades are set in motion.While in some embodiments these multiple pathways may converge on asingle signaling molecule (e.g., IFNγ), the overall cytotoxic effect isunexpectedly increased because of the overall magnitude of signalingmolecules driving a cytotoxic endpoint. As a non-limiting example, inseveral embodiments an NKG2D is coupled to a CD8a hinge followed by aCD16 transmembrane-intracellular signaling domain and a 4-1BB signalingdomain. In several embodiments, this construct further comprises a 2B4signaling domains. In several embodiments, such a chimeric receptor hasthe nucleic acid sequence of SEQ ID NO. 53. In some embodiments, thechimeric receptor may vary from SEQ ID NO. 53, but remains, depending onthe embodiment, at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, or at least 95% homologous with SEQ ID NO. 53. Inadditional embodiments, the NKG2D-CD8a-CD16IC/TM construct furthercomprises a NKp80 signaling domain. In several embodiments, such aconstruct further comprises a GS3 linker between the 4-1BB and NKp80domains. In several embodiments, such a chimeric receptor has thenucleic acid sequence of SEQ ID NO. 54. In some embodiments, thechimeric receptor may vary from SEQ ID NO. 54, but remains, depending onthe embodiment, at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, or at least 95% homologous with SEQ ID NO. 54.

In still additional embodiments, certain components of a chimericreceptor can be replaced with one or more additional subunits that leadto enhanced efficacy (e.g., activation or cytotoxicity of NK cells). Forexample, in one embodiment, a CD16 intracellular signaling domain can bereplaced with a quad-repeat of DAP10 (e.g., 4xDAP10). In an additionalembodiment, a CD16 intracellular signaling domain can be replaced with aZap70 subunit. Certain such embodiments lead to unexpectedly enhanced NKcell cytotoxicity.

In several additional embodiments, the effector domain comprises one ormore consensus hemi-ITAM sequences to enhance the transduction ofactivation signaling upon ligand binding. In additional embodiments, theinclusion of a GS linker between the signaling domains of 4-1BB, CD16,NCR1, NCR2 and/or NCR3 enhances signal transduction. Moreover, inseveral embodiments one or both of CD3ζ and FcRγ are additionallyexpressed along with the chimeric receptors described herein (either onthe same or a separate construct), which results in unexpectedlyenhanced signal transduction, and thus an unexpected increase incytotoxic effects of the NK cells. Depending on the embodiment, theengineered expression of one or more of CD3ζ and FcRγ supplementsendogenous expression of these molecules by NK cells, thereby furtherenhancing the signaling and ultimate cytotoxic potency of the NK cells.

Optionally, depending on the embodiment, any of the polynucleotidesdisclosed herein may also encode truncations and/or variants of one ormore of the constituent subunits of a chimeric receptor, yet retaintheir ability to direct NK cells to target cells and in severalembodiments unexpectedly enhance cytotoxicity upon binding. In addition,any of the polynucleotides disclosed herein may also optionally includecodon-optimized nucleotide sequences encoding the various constituentsubunits of a chimeric receptor. As used herein, the terms “fragment”and “truncated” shall be given their ordinary meaning and shall alsoinclude N- and C-terminal deletion variants of proteins.

The polynucleotides encoding the chimeric receptors described herein maybe inserted into vectors to achieve recombinant protein expression in NKcells. In one embodiment, the polynucleotide is operably linked to atleast one regulatory element for the expression of the chimericreceptor. In specific embodiments, transcriptional regulatory elementsheterologous, such as, for example an internal ribosome entry site(IRES) or enhancer element, to the peptides disclosed herein areemployed to direct the transcription of the chimeric receptor. In someembodiments, the polynucleotide comprises one or more cytosolic proteasecleavage sites. In some embodiments, the cleavage site is recognized andcleaved by a cytosolic protease. In some embodiments, this cleavage siteis selected from the group comprising a T2A cleavage site, a P2Acleavage site, an E2A cleavage site, and an F2A cleavage site. Dependingon the embodiment, the various constituent parts of a chimeric receptorcan be delivered to an NK cell in a single vector, or alternatively inmultiple vectors. In some embodiments, a chimeric receptor construct isdelivered in a single vector, while another factor that enhancesefficacy of the chimeric receptor, such as mbIL15, is delivered in aseparate vector. In several embodiments, a chimeric receptor and afactor that enhances efficacy of the chimeric receptor (e.g., mbIL15),is delivered in a single vector. Regardless of the number of vectorsused, any polynucleotide may optionally include a tag sequence, allowingidentification of the presence of NK cells expressing the construct. Forexample, in several embodiments a FLAG tag (DYKDDDDK, SEQ ID NO. 55) isused. Also available are other tag sequences, such as a polyhistidinetag (His-tag) (HHHHHH, SEQ ID NO. 56), HA-tag or myc-tag (EQKLISEEDL;SEQ ID NO: 57). Alternatively, green fluorescent protein, or otherfluorescent moiety, is used. Combinations of tag types can also be used,to individually recognize sub-components of a chimeric receptor.

In several embodiments, the polynucleotide encoding the chimericreceptor is an mRNA that may be introduced into NK cells byelectroporation. In another embodiment, the vector is a virus,preferably a retrovirus, which may be introduced into NK cells bytransduction. In several embodiments, the vector is a Murine Stem CellVirus (MSCV). In additional embodiments, other vectors may be used, forexample lentivirus, adenovirus, adeno-associated virus, and the like maybe used. In several embodiments, non-HIV-derived retroviruses are used.The vector chosen will depend upon a variety of factors, including,without limitation, the strength of the transcriptional regulatoryelements and the cell to be used to express a protein. The vector can bea plasmid, phagemid, cosmid, viral vector, phage, artificial chromosome,and the like. In additional embodiments, the vectors can be episomal,non-homologously, or homologously integrating vectors, which can beintroduced into the appropriate cells by any suitable means(transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphate-precipitation, direct microinjection,etc.) to transform them. Other approaches to induce expression ofchimeric receptors in NK cells are used in several embodiments,including for example, the SV40 early promoter region, the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus, theherpes thymidine kinase promoter, the regulatory sequences of themetallothionein gene, an adenovirus (ADV) promoter, a cytomegalovirus(CMV) promoter, the bovine papilloma virus (BPV) promoter, the parovirusB19p6 promoter, the beta-lactamase promoter, the tac promoter, thenopaline synthetase promoter region or the cauliflower mosaic virus 35SRNA promoter, the promoter of ribulose biphosphate carboxylase, the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK(phosphoglycerol kinase) promoter, the synthetic MND promoter containingthe U3 region of a modified MoMuLV LTR with the myeloproliferativesarcoma virus enhancer, and the alkaline phosphatase promoter.

Natural killer cells may be engineered to express the chimeric receptorsdisclosed herein. Chimeric receptor expression constructs may beintroduced into NK cells using any of the techniques known to one ofskill in the art. In one embodiment, the chimeric receptors aretransiently expressed in the NK cells. In another embodiment, thechimeric receptors are stably expressed in NK cells. In an additionalembodiment, the NK cells are autologous cells. In yet anotherembodiment, the NK cells are donor-derived (allogeneic) cells.

Further provided herein are methods of treating a subject having canceror an infectious disease comprising administering to the subject acomposition comprising NK cells engineered to express a chimericreceptor as disclosed herein, the chimeric receptor designed to target amarker or ligand expressed differentially on the damaged or diseasedcells or tissue (e.g., expressed to a different degree as compared to anormal cell or tissue). As used herein, the terms “express”, “expressed”and “expression” be given their ordinary meaning and shall refer toallowing or causing the information in a gene or polynucleotide sequenceto become manifest, for example producing a protein by activating thecellular functions involved in transcription and translation of acorresponding gene or DNA sequence. The expression product itself, e.g.,the resulting protein, may also be said to be “expressed” by the cell.An expression product may be characterized as intracellular,extracellular or transmembrane. The term “intracellular” shall be givenits ordinary meaning and shall refer to inside a cell. The term“extracellular” shall be given its ordinary meaning and shall refer tooutside a cell. The term “transmembrane” shall be given its ordinarymeaning and shall refer to at least a portion of a polypeptide isembedded in a cell membrane. The term “cytoplasmic” shall be given itsordinary meaning and shall refer to residing within the cell membrane,outside the nucleus. As used herein, the terms “treat,” “treating,” and“treatment” in the context of the administration of a therapy to asubject shall be given their ordinary meaning and shall refer to thebeneficial effects that a subject derives from a therapy. In certainembodiments, treatment of a subject with a genetically engineeredcell(s) described herein achieves one, two, three, four, or more of thefollowing effects, including, for example: (i) reduction or ameliorationthe severity of disease or symptom associated therewith; (ii) reductionin the duration of a symptom associated with a disease; (iii) protectionagainst the progression of a disease or symptom associated therewith;(iv) regression of a disease or symptom associated therewith; (v)protection against the development or onset of a symptom associated witha disease; (vi) protection against the recurrence of a symptomassociated with a disease; (vii) reduction in the hospitalization of asubject; (viii) reduction in the hospitalization length; (ix) anincrease in the survival of a subject with a disease; (x) a reduction inthe number of symptoms associated with a disease; (xi) an enhancement,improvement, supplementation, complementation, or augmentation of theprophylactic or therapeutic effect(s) of another therapy. Administrationcan be by a variety of routes, including, without limitation,intravenous, intra-arterial, subcutaneous, intramuscular, intrahepatic,intraperitoneal and/or local delivery to an affected tissue. Doses of NKcells can be readily determined for a given subject based on their bodymass, disease type and state, and desired aggressiveness of treatment,but range, depending on the embodiments, from about 10⁵ cells per kg toabout 10¹² cells per kg (e.g., 10⁵⁻10⁷, 10⁷⁻10¹⁰, 10¹⁰⁻10¹² andoverlapping ranges therein). In one embodiment, a dose escalationregimen is used. In several embodiments, a range of NK cells isadministered, for example between about 1×10⁶ cells/kg to about 1×10⁸cells/kg. Depending on the embodiment, various types of cancer orinfection disease can be treated. Various embodiments provided forherein include treatment or prevention of the following non-limitingexamples of cancers including, but not limited to, acute lymphoblasticleukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma,Kaposi sarcoma, lymphoma, gastrointestinal cancer, appendix cancer,central nervous system cancer, basal cell carcinoma, bile duct cancer,bladder cancer, bone cancer, brain tumors (including but not limited toastrocytomas, spinal cord tumors, brain stem glioma, craniopharyngioma,ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma),breast cancer, bronchial tumors, Burkitt lymphoma, cervical cancer,colon cancer, chronic lymphocytic leukemia (CLL), chronic myelogenousleukemia (CML), chronic myeloproliferative disorders, ductal carcinoma,endometrial cancer, esophageal cancer, gastric cancer, Hodgkin lymphoma,non-Hodgkin lymphoma, hairy cell leukemia, renal cell cancer, leukemia,oral cancer, nasopharyngeal cancer, liver cancer, lung cancer (includingbut not limited to, non-small cell lung cancer, (NSCLC) and small celllung cancer), pancreatic cancer, bowel cancer, lymphoma, melanoma,ocular cancer, ovarian cancer, pancreatic cancer, prostate cancer,pituitary cancer, uterine cancer, and vaginal cancer.

Further, various embodiments provided for herein include treatment orprevention of the following non-limiting examples of infectious diseasesincluding, but not limited to, infections of bacterial origin mayinclude, for example, infections with bacteria from one or more of thefollowing genera: Bordetella, Borrelia, Brucella, Campylobacter,Chlamydia and Chlamydophila, Clostridium, Corynebacterium, Enterococcus,Escherichia, Francisella, Haemophilus, Helicobacter, Legionella,Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas,Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus,Treponema, Vibrio, and Yersinia, and mutants or combinations thereof. Inseveral embodiments, methods are provided to treat a variety to treatviral infections, such as those caused by one or more viruses, such asadenovirus, Coxsackievirus, Epstein-Barr virus, hepatitis a virus,hepatitis b virus, hepatitis c virus, herpes simplex virus, type 1,herpes simplex virus, type 2, cytomegalovirus, ebola virus, humanherpesvirus, type 8, HIV, influenza virus, measles virus, mumps virus,human papillomavirus, parainfluenza virus, poliovirus, rabies virus,respiratory syncytial virus, rubella virus, and varicella-zoster virus.

In some embodiments, also provided herein are nucleic acid and aminoacid sequences that have homology of at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% (and ranges therein) as compared with the respectivenucleic acid or amino acid sequences of SEQ ID NOS. 1-68 and that alsoexhibit one or more of the functions as compared with the respective SEQID NOS. 1-68: including but not limited to, (i) enhanced proliferation,(ii) enhanced activation, (iii) enhanced cytotoxic activity againstcells presenting ligands to which NK cells harboring receptors encodedby the nucleic acid and amino acid sequences bind, (iv) enhanced homingto tumor or infected sites, (v) reduced off target cytotoxic effects,(vi) enhanced secretion of immunostimulatory cytokines and chemokines(including, but not limited to IFNg, TNFa, IL-22, CCL3, CCL4, and CCL5),(vii) enhanced ability to stimulate further innate and adaptive immuneresponses, and (viii) combinations thereof.

Additionally, in several embodiments, there are provided amino acidsequences that correspond to any of the nucleic acids disclosed herein,while accounting for degeneracy of the nucleic acid code. Furthermore,those sequences (whether nucleic acid or amino acid) that vary fromthose expressly disclosed herein, but have functional similarity orequivalency are also contemplated within the scope of the presentdisclosure. The foregoing includes mutants, truncations, substitutions,or other types of modifications.

There are provided for herein, according to several embodiments,polynucleotides encoding chimeric receptors, comprising an extracellularreceptor domain, wherein the extracellular receptor domain comprises apeptide that binds native ligands of Natural Killer Group 2 member D(NKG2D), wherein the peptide that binds native ligands of NKG2D is afragment of NKG2D, an effector domain comprising a transmembrane regionand an intracellular signaling domain. In several embodiments, thefragment of NKG2D is encoded by a polynucleotide comprising the sequenceof SEQ ID NO. 2 or 3 or 68, or functional equivalent thereof. In severalembodiments, the polynucleotide encodes an effector domain comprisingCD16. In several embodiments, the polynucleotide encodes an effectordomain comprising NCR1. In several embodiments, the polynucleotideencodes an effector domain comprising NCR2. In several embodiments, thepolynucleotide encodes an effector domain comprising NCR3. In someembodiments, the polynucleotide encodes an additional effector domainportion comprising 4-1BB. In several embodiments, the polynucleotideencodes a chimeric receptor made up of NKG2D and CD16. In severalembodiments, the polynucleotide encodes a chimeric receptor made up ofNKG2D and NCR1. In several embodiments, the polynucleotide encodes achimeric receptor made up of NKG2D and NCR2. In additional embodiments,the polynucleotide encodes a chimeric receptor made up of NKG2D coupledto CD16 and optionally 4-1BB. In several embodiments, CD16 is replacedby NCR1, and in some embodiments, by NCR2, or even NCR3, depending onthe embodiment. In several embodiments, the effector domain furthercomprises a GS linker between, for example, 4-1BB and one of CD16, NCR1,NCR2, or NCR3.

In several embodiments, the extracellular receptor domain furthercomprises a hinge region. In several embodiments, the hinge regioncomprises CD8a. However, in additional embodiments, the hinge regionfurther comprises one or more linkers, which in some embodiments,comprise GS9, CD8a/GS3, truncated CD8a, GS3, and the like.

In several embodiments, the extracellular receptor domain furthercomprises a CD8a signal peptide. In several embodiments, the effectordomain comprises one or more hemi-ITAM sequences. In severalembodiments, the chimeric receptor does not comprise DNAX-activatingprotein 10 (DAP10). In several embodiments, the chimeric receptor doesnot comprise an ITAM motif, but rather employs an alternative signalingregion, such as an ITSM, hemi-tam or other co-stimulatory region.

In one embodiment, there is provided a polynucleotide encoding achimeric receptor comprising an extracellular receptor domain, whereinthe extracellular receptor domain comprises a peptide that binds nativeligands of Natural Killer Group 2 member D (NKG2D), wherein the peptidethat binds native ligands of NKG2D is a fragment of NKG2D, atransmembrane region, wherein the transmembrane region comprises CD8a,and an effector domain, wherein the effector domain comprises 4-1BB andCD3 zeta, wherein the polynucleotide is co-expressed with an additionalconstruct encoding membrane-bound interleukin 15 (mbIL15).

There is also provided in several embodiments, a polynucleotide encodinga chimeric receptor comprising an extracellular receptor domain, whereinthe extracellular receptor domain comprises a peptide that binds nativeligands of Natural Killer Group 2 member D (NKG2D), wherein the peptidethat binds native ligands of NKG2D is a fragment of NKG2D, atransmembrane region, wherein the transmembrane region comprises CD8a,and an effector domain, wherein the effector domain comprises 4-1BB andthe intracellular domain of 2B4 or DAP10. The polynucleotide encoding achimeric receptor as described herein comprises a second peptide thatbinds native ligands of NKG2D. In several embodiments, the nativeligands of NKG2D include, but are not limited to, MICA, MICB, ULBP1,ULBP2, ULBP3, ULBP4, ULBP5 or ULBP6. In several embodiments, the portionof the chimeric receptor that binds native ligands of NKG2D has at least80% homology to SEQ ID NO: 1, 2, 3, or 68.

In several embodiments, the provided polynucleotide is an mRNA. In someembodiments, the polynucleotide is operably linked to at least oneregulatory element for the expression of the chimeric receptor. As usedherein, the terms “nucleic acid,” “nucleotide,” and “polynucleotide”shall be given their ordinary meanings and shall includedeoxyribonucleotides, deoxyribonucleic acids, ribonucleotides, andribonucleic acids, and polymeric forms thereof, and includes eithersingle- or double-stranded forms. Nucleic acids include naturallyoccurring nucleic acids, such as deoxyribonucleic acid (“DNA”) andribonucleic acid (“RNA”) as well as nucleic acid analogs. Nucleic acidanalogs include those which include non-naturally occurring bases,nucleotides that engage in linkages with other nucleotides other thanthe naturally occurring phosphodiester bond or which include basesattached through linkages other than phosphodiester bonds. Thus, nucleicacid analogs include, for example and without limitation,phosphorothioates, phosphorodithioates, phosphorotriesters,phosphoramidates, boranophosphates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs),locked-nucleic acids (LNAs), and the like. As used herein, the term“operably linked,” for example in the context of a regulatory nucleicacid sequence being “operably linked” to a heterologous nucleic acidsequence, shall be given its ordinary meaning and shall mean that theregulatory nucleic acid sequence is placed into a functionalrelationship with the heterologous nucleic acid sequence. In the contextof an IRES, “operably linked to” refers to a functional linkage betweena nucleic acid sequence containing an internal ribosome entry site and aheterologous coding sequence initiation in the middle of an mRNAsequence resulting in translation of the heterologous coding sequence.As used herein, the term “vector” shall be given its ordinary meaningand shall refer to a vehicle by which a DNA or RNA sequence (e.g., aforeign gene) can be introduced into a genetically engineered cell, soas to transform the genetically engineered cell and promote expression(e.g., transcription and/or translation) of the introduced sequence.Vectors include viruses, plasmids, phages, etc. The term “chimericreceptor” as used herein shall be given its ordinary meaning and shallrefer to a cell-surface receptor comprising at least two polypeptidedomains not naturally found together on a single protein. The term“chimeric receptor complex” as used herein refers to a firstpolypeptide, which may comprise at least two polypeptide domains in acombination that are not naturally found together on a single protein,which first polypeptide is associated with a second polypeptide, forexample, an adaptor polypeptide, a signaling molecule, or a stimulatorymolecule. Additional terms relating to generation and use of chimericreceptors as disclosed here are readily understood by one of ordinaryskill in the art and can also be found in International Publication WO2014/117121 and U.S. Pat. No. 7,994,298, each of which are incorporatedby reference in their entirety herein.

Additionally provided, according to several embodiments, is a vectorcomprising the polynucleotide encoding any of the polynucleotidesprovided for herein, wherein the polynucleotides are optionallyoperatively linked to at least one regulatory element for expression ofa chimeric receptor. In several embodiments, the vector is a retrovirus.

Further provided herein are engineered natural killer cells comprisingthe polynucleotide, vector, or chimeric receptors as disclosed herein.In several embodiments, these NK cells are suitable for use in thetreatment of prevention of disease, such as, for example, cancer and/orinfectious disease.

EXAMPLES Methods

The following experimental methods and materials were used in thenon-limiting experimental examples disclosed below.

Cell Lines and Culture Conditions

The human acute lymphoblastic leukemia cell line REH, human osteosarcomacell line U-2 OS and human embryonic kidney fibroblast 293T (HEK 293T)cells were obtained from the American Type Culture Collection (ATCC;Manassas, Va). REH cells were maintained and grown in Roswell ParkMemorial Institute series 1640 (RPMI-1640; Gibco, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, Utah) and1% penicillin-streptomycin. Both HEK 293T and U-2 OS cells weremaintained and grown in Dulbecco's modified Eagles Medium (DMEM;Hyclone) supplemented with 10% FBS and 1% penicillin-streptomycin. Allmammalian cells were incubated at 37° C. with 5% CO2.

DNA Plasmids

A DNA plasmid containing the chimeric receptor NKG2D-DAP10-CD3ζ was madeas previously described (see Chang et al. Cancer Research, Vol. 73(6):2013). Splicing by overlapping extension polymerase chain reaction(SOE-PCR) was used to fuse the individual domains forming theNKG2D-41BB-CD3ζ construct. That construct was then inserted into theMurine Stem Cell Virus (MSCV) retroviral vector (FIG. 3A). Theconstructs for NKG2D-CD16 and NKG2D-CD16-41BB were codon optimized andinserted into the MSCV vector (FIG. 3B) by GenScript (Nanjing, China).The sequences of the constructs were verified by DNA sequencing.

Expansion of Human NK Cells

Human peripheral blood mononuclear cells (PBMCs) were obtained by Ficolldensity centrifugation of blood samples from healthy adult donors. Toexpand the NK cells, PBMCs were cultured with K562 genetically modifiedwith membrane bound IL-15 and 4-1BB ligand (K562-mb15-41BBL). Cells werecultured in Stem Cell Growth Medium (SCGM; Cell Genix, Freiburg,Germany) supplemented with 40 IU of IL-2/ml every two days.

After 7 days of culture, NK cells were T-cell depleted using anti-CD3Dynabeads (Invitrogen, Carlsbad, Calif.). NK cells were then cultured inSCGM supplemented with 40-200 IU of IL-2/ml every two days.

Production of Retrovirus and Transduction of NK Cells

Production of retrovirus was carried out by transiently transfecting HEK293T cells with retroviral packaging plasmids. HEK 293T cells were firstseeded to a concentration of 2.5×10⁶ cells in 12 ml of DMEM 18 hoursbefore the transfection. The cells were then transfected with 3.5 μg ofMSCV vector containing the respective NKG2D chimeric receptors(non-limiting constructs are illustrated schematically in FIGS. 1B-1Cand 2A-2B), 3.5 μg of pEQ-PAM3, and 3.0 μg of pRDF. For control, emptyMSCV vector containing GFP was used. X-tremeGENE 9 DNA TransfectionReagent (Roche, Basel, Switzerland) was used for the transfection. DMEMwas replaced with conditioned RPMI-1640 24 hours after the transfection.

Transduction of NKG2D chimeric receptor transgene into NK cells was done18 hours after the changing of media. NK cells were first suspended at aconcentration of 0.25×10⁶ cells in 2 ml of conditioned RPMI-1640. Cellswere subsequently seeded into RetroNectin (TaKaRa, Otsu, Japan) coatedtubes. RPMI-1640 containing the retrovirus (virus supernatant) washarvested from the HEK 293T cell cultures and fresh conditioned mediumwas added back to the cultures. The viral supernatant was supplementedwith 200 IU of IL-2/ml and 3 ml of the viral supernatant was dispensedinto each RetroNectin coated tubes (containing the seeded NK cells). Inaccordance with certain embodiments of producing NK cells, seeded NKcells were transduced six times, once every 12 hours with fresh viralmedia. Transduced NK cells were then harvested 48 hours after the lasttransduction, and cultured in SCGM with the addition of 200 IU ofIL-2/ml every two days. The transduced NK cells were used forexperiments 14 to 28 days after expansion.

Detection of Chimeric Receptor Expression by Flow Cytometry

Transduced NK cells were washed once with phosphate-buffered salinecontaining albumin, and 2 μl of rabbit serum was added. The cells werethen stained with peridinin chlorophyll (PerCP)-conjugated anti-humanNKG2D antibody (clone 149810; R&D Systems, Minneapolis, USA) for 10minutes in the dark. For controls, the transduced NK cells were stainedwith the respective PerCP-conjugated IgG isotype antibody. All NK cellswere washed again and fixed with 300 μl 0.5% formaldehyde beforeanalysis using Accuri C6 flow cytometer (BD, Franklin Lakes, N.J.). Datawas analyzed using a paired t-test.

Cytotoxicity Assays

REH cells were stained with calcein AM red-orange (Thermo FisherScientific, Waltham, Mass.). REH cells were seeded into a 96-well roundbottom plate (CoStar, Corning, N.Y.). Transduced NK cells were thenadded at various effector: target (E:T) ratio. The cell cultures wereincubated for four hours at 37° C. and 5% CO₂. Stained viable targetcells were counted using the Accuri C6 flow cytometer. U-2 OS cells wereseeded into 96-well flat bottom white plate (Costar) and incubated forfour hours. Transduced NK cells was then added according to differentE:T ratios. Cell cultures were then incubated for another four hours.Prior to analysis, Bright-Glo substrate (Promega, Madison, Wis.) wasadded to the cells. Intensity of luminescence from viable target cellswas measured using FLx800 Fluorescence Reader (Bio Tek, Winooski, Vt.).Differences between intensity of luminescence and control were convertedto percentage cytotoxicity.

Interferon Gamma (IFNγ) Production Assay

To determine the amount of IFNγ produced by the NK cells, effector andtarget cells were first cultured with (E:T of 1:1) or without REH in a96-well round bottom plate. Cells were incubated for one hour before theaddition of GolgiPlug (brefeldin A; BD Biosciences). After another 5hours of culture, cells were labeled with phycoerythrin (PE)-conjugatedanti-human CD56 antibody (clone MY31, BD Biosciences). Cells werepermeabilized using a proprietary permeabilization reagent and incubatedfor 40 minutes in the dark. The cells were then washed with aproprietary wash buffer. Intracellular IFNγ was detected withallophycocyanin (APC)-conjugated IFNγ antibody (clone 25723.11; BDBiosciences) for 45 minutes. The cells were then fixed and analyzedusing Accuri C6 flow cytometer.

Example 1—CD3-Zeta Containing NKG2D Constructs

As disclosed herein, various constructs comprising NKG2D and/or NKG2Dvariants coupled with various transmembrane and/or signaling domains areprovided. The present experiment was conducted to evaluate theexpression and cytotoxic activity of constructs comprising CD3-zetasignaling domains. Two CD3-zeta constructs were prepared and testedaccording to the methods and materials described above. Depending on theconstruct, the methods used can be readily adjusted to account forvariations required for generating, expressing and testing a construct.The two constructs were NKG2D-DAP10-CD3ζ and NKG2D-41BB-CD3ζ. Forreference FIG. 1A schematically depicts an endogenous NKG2D. In NKcells, ionic interactions between the transmembrane region of NKG2Dallow association with its adaptor protein DAP10 (Wu et al., 1999). Uponligand binding, NKG2D signals are transduced through the signalingmotif, YxNM, found on DAP10. CD3ζ transduce signals through itsimmunoreceptor tyrosine-based activation motif (ITAM; Lanier, 2008). Thetwo experimental constructs are illustrated schematically in FIGS. 1Band 1C, respectively. FIG. 1B shows NKG2D-DAP10-CD3ζ, with signalingoccurring through both the YxNM and ITAM motifs. FIG. 1C shows theNKG2D-41BB-CD3ζ construct, which employs a CD8a hinge region as atransmembrane domain and 4-1BB and CD3ζ as signaling domains.

The ability of NK cells to effectively express these constructs wasfirst assessed. NK cells expanded from PBMC of healthy adult donors weretransduced with one of the two chimeric receptors. Mock-transduced NKcells were used as control (transduced with empty MSCV vector containingGFP only). The presence and relative abundance of the chimeric receptorswere determined through staining the NK cells with a Per-CP conjugatedanti-NKG2D antibody. FIG. 4A depicts representative flow cytometry datarelated to the percentage of NKG2D-positive NK cells after transductionwith Mock (left panel), NKG2D-DAP10-CD3ζ (center panel) orNKG2D-41BB-CD3ζ (right panel) constructs. Mock transduced NK cell showedno NKG2D expression with the antibody used (which does not showingstaining above an isotype-matched non-reactive antibody, despite thenaturally high NKG2D expression on activated NK cells), while just under60% of cells transduced with the NKG2D-DAP10-CD3ζ construct exhibitedNKG2D expression above the isotype-matched non-reactive antibodycontrol, and over 80% of NK cells transduced with the NKG2D-41BB-CD3ζ.Pooled data for the percentage of NKG2D positive NK cells from alldonors is shown in FIG. 4B. Both engineered NKG2D constructs result insubstantial gain in NKG2D expression compared to Mock, though there isnot a significant difference between the percent expression of the twoconstructs. FIG. 4C depicts expression data based on Mean FluorescenceIntensity (MFI), which represents, within the population expressing theNKG2D construct, the degree to which that cell expresses the construct(e.g., multiple copies of the construct per cell would yield a greaterMFI). By this measure, the expression of the NKG2D-41BB-CD3ζ issignificantly greater than that of the NKG2D-DAP10-CD3ζ construct.

Collectively, these data demonstrate that, in accordance with severalembodiments disclosed herein, engineered constructs can successfully beexpressed on NK cells. In several embodiments, enhanced expression ofthe construct can be achieved by repeated transduction of the NK cellswith a particular construct. In several embodiments, the components ofthe constructs can be delivered to a cell in a single vector, oralternatively using multiple vectors. Depending on the embodiment, theconstruct itself may lead to enhanced expression, for example a linearor head to tail construct may yield increased expression because of alesser degree of in-cell assembly that a multiple subunit constructrequires.

Further to successfully expressing NKG2D constructs on NK cells,effective signaling of the NK cells is required to act on target cells.To evaluate the potency of the two populations of transduced NK cells,cytotoxicity assays were performed using to cell lines that aresensitive to NK cell activity, REH (suspension cells) and U-2 OS(adherent cells). Data summarizing the percentage cytotoxicity of thedifferent groups of NK cells against REH cells and across independentdonors at two E:T ratios are shown in FIGS. 5A-5C (error bars representstandard deviation; all experiments are done in triplicates; n=3(P<0.001)). As depicted in FIGS. 5A-5C, NK cells expressing either NKG2Dchimeric receptor (NKG2D-DAP10-CD3ζ shown with an arrow labeled (a) andNKG2D-41BB-CD3ζ shown with an arrow labeled (b)) had a significantlyhigher cytotoxicity against REH for all three donors as compared to mockNK cells (shown with an arrow labeled (c)). The mean percentagecytotoxicity of NKG2D-DAP10-CD3ζ-expressing NK cells was 91.8%±5.8% (1:1E:T ratio) and 83.9%±5.6% (1:2 E:T ratio). Those NK cells transducedwith NKG2D-41BB-CD3ζ showed similar potencies −87.4%±6.1% at a 1:1 E:Tratio and 76.2%±4.8% at a 1:2 E:T ratio. Chimeric receptor-expressing NKcells also showed elevated cytotoxicity against U-2 OS when compared tomock-transduced NK cells (See FIGS. 6A-6C, FIG. 6A depictsNKG2D-DAP10-CD3ζ shown with an arrow labeled (a), FIG. 6B depictsNKG2D-41BB-CD3ζ shown with an arrow labeled (b) and FIG. 6C depicts mockNK cells shown with an arrow labeled (c)).

These data provide evidence that NK cells can not only be engineered toexpress chimeric receptor constructs, but those cells that express thechimeric receptors are able to be activated and successfully generateenhanced cytotoxic effects against target cells. Importantly, these dataalso show that there is only a slight decrease in the potency of thecells when in the presence of a greater number of target cells (doubledin this experiment). This suggests that the desired cytotoxic effects ofthe engineered NK cells can still be realized, even when the NK cellsare present in smaller numbers vis-à-vis target cells, as would likelybe the case in clinical use. Moreover, these data indicate that,according to some embodiments, a lesser density or degree of chimericreceptor expression on a given NK cell does not necessarily result incoordinately reduced cytotoxic effects, and can be associated with anunexpected efficacy of the NK cells in view of their lesser constructexpression. Additionally, these data embody the unexpectedly enhancedcytotoxicity that is achieved according to several embodiments. Whilenon-engineered NK cells are cytotoxic, and express a significant amountof NKG2D upon activation, it is unexpected that the engineered cellsdisclosed herein can push the cytotoxic effects significantly beyondwhat can be considered an already elevated ceiling (e.g., native NK cellcytotoxicity).

Further to the cytotoxicity data, the mechanism by which the NK cellsare exerting these effects was examined, by evaluating the production ifinterferon-gamma (IFNγ) by the NK cells expressing the various NKG2Dconstructs. IFNγ is a key cytokine produced and released by NK cells(typically during an innate immune response) that recruits macrophagesand has immunostimulatory effects. FIG. 7A shows the relative amount ofIFNγ production (measured by MFI) in Mock (left panel),NKG2D-DAP10-CD3ζ-expressing NK cells (center panel), andNKG2D-41BB-CD3ζ-expressing NK cells (right panel) with or withoutstimulation by REH cells. NK cells were stained by APC-conjugatedanti-IFNγ antibody for intracellular IFNγ. Data was analyzed by paired ttest. These data show that each of the three groups of NK cells wereobserved to have a similar level of IFNγ production without stimulation,with an increase observed after stimulation by REH cells. As providedfor in several embodiments, engineered NK cells expressing NKG2Dconstructs can lead to robust cytokine production. The presence of atarget cell (here, REH cells) to which the engineered NK cells respondssets into motion the biochemical cascade which leads to IFNγ productionand ultimately cytotoxic effects. As shown in FIG. 7A, theNKG2D-41BB-CD3ζ-expressing NK cells show a robust production of IFNγ inthe presence of stimulatory REH cells. Interestingly, theNKG2D-DAP10-CD3ζ-expressing NK cells failed to show a similar degree ofresponse. This is further demonstrated in FIG. 7B, where levels of IFNγbetween different groups of NK cells after stimulation with REH cells(median values were represented; data was analyzed by unpaired t test)are evaluated. All IFNγ experiments were conducted in triplicates, withthree independent donors, n=9. FIG.7B shows that IFNγ production byNKG2D-DAP10-CD3ζ-expressing NK cells was not significantly differentfrom mock-transduced NK cells. In contrast, theNKG2D-41BB-CD3ζ-expressing NK cells show a significant increase in IFNγproduction as compared to mock-transduced NK cells. These data areinteresting because they demonstrate that, as discussed herein,signaling by a chimeric receptor in response to ligand binding is anessential step in generating cytotoxic effects against a target cell ofinterest. However, there is not a singular pathway through which thevarious constructs signal, as NK cells transduced with two differentchimeric receptors both exhibit relatively similar cytotoxicity, butwithout mirroring levels of IFNγ production. Thus, according to someembodiments, constructs are provided that achieve cytotoxic effectsthrough an elevated production of IFNγ, or other immunostimulatorycytokine, as compared to normal NK cells. However, in severalembodiments, increased production of IFNγ is not necessarily achieved ordetected, rather another immunostimulatory pathway can be exploited by agiven chimeric construct to achieve elevated cytotoxic effects.

Example 2—CD16 and CD16-4-1BB Containing NKG2D Constructs

Additional constructs were generated to evaluate expression,cytotoxicity and cytokine production. As provided for herein, severalembodiments relate to constructs comprising a truncated NKG2D (in someembodiments codon optimized), that employ a CD16 transmembrane and/orsignaling domain. The constructs generated for evaluation in thisexperiment are schematically shown in FIGS. 2A-2B, which show thestructure of A) NKG2D-CD16 and B) NKG2D-CD16-41BB chimeric receptors.Both chimeric receptors rely on the transmembrane region of CD16 toassociate with either CD3ζ or FcRγ. The plasmids used to generate theseconstructs are shown in FIG. 3B. As discussed above, in severalembodiments, the constructs employed rely on endogenous expression ofCD3ζ or FcRγ, however, in several embodiments the plasmid encoding thechimeric receptor (or a separate plasmid) is configured to elevateexpression of CD3ζ and/or FcRγ by the NK cell, thereby enhancing thepotency of the cells.

As above, expression levels of the constructs were evaluated. FIG. 8Adepicts representative flow cytometry data for mock (left panel),NKG2D-DAP10-CD3ζ-expressing NK cells (center panel), andNKG2D-CD16-expressing NK cells (Experiments were conducted using cellsfrom three independent donors represented by different symbols. Data wasanalyzed by paired t test). FIG. 8B shows summary data relating to thepercentage of cells that that express NKG2D (and hence the constructs).As expected, mock-transfected NK cells show low levels of NKG2Dexpression with the antibody used. In contrast, both of the engineeredconstructs exhibited significantly enhanced expression, withNKG2D-CD16-transduced NK cells expressing 35.8%±6.9% greater expressionas compared to mock-transduced NK cells. Additionally, as evaluated byMFI (FIG. 8C), NKG2D-CD16-transduced NK cells also exhibited increasedexpression of the construct. These data are important to demonstratethat the constructs can effectively be introduced into NK cells and areexpressed.

Having established expression of the constructs, their ability toexhibit cytotoxic effects was evaluated. As discussed above, NK cellsfrom three donors were tested for cytotoxic effects against REH cellsand U-2 OS cells, each at three E:T ratios (all experiments were done intriplicate, n=3). Interestingly, the enhanced expression of theNKG2D-CD16 construct as compared to mock NK cells did not result inincreased cytotoxicity (see FIG. 9A-9C, error bars represent standarddeviations). As with the prior example, NKG2D-DAP10-CD3ζ-expressing NKcells (shown with an arrow labeled (a)) did exhibit an increasedcytotoxicity. With respect to cytotoxicity against U-2 OS cells, theNKG2D-CD16 (shown with an arrow labeled (b)) did exhibit an increasedcytotoxicity as compared to mock NK cells (shown with an arrow labeled(c)) (see FIGS. 10A-10C). These data indicate that the degree ofcytotoxic impact on a particular given target cell type may vary withthe NK construct used. In some embodiments, a particular construct maynot be as effective, however, in several embodiments, combinations ofpopulations of NK cells can be used and exhibit synergistic effects. Inother words, a population of NK cells, with a portion expressingNKG2D-CD16 and a portion expressing NKG2D-DAP10-CD3ζ (or othercombination of any of the constructs disclosed herein), may exhibitunexpectedly enhanced cytotoxicity as compared to either sub-populationalone.

Interferon-γ production was measured next, in order to confirm themechanism of action of the transfected NK cells. The NK cells expressingthe various constructs were either stimulated by REH cells, or not, andthe production of IFNγ was measured. These data are shown in FIG. 11(data was analyzed by paired t test). All groups of NK cells had similarlevel of IFNγ without stimulation, and an increase after incubation withREH cells. The NKG2D-CD16-expressing NK cells exhibited an increase inIFNγ production of 634±211 MFI, which was greater than the increaseexhibited by the mock-transduced NK cells (423±70 MFI). However, theincrease was lower than that observed for NKG2D-DAP10-CD3ζ-expressing NKcells, which increased 2041±411 MFI. In line with data, according toseveral embodiments the production of IFNγ is correlated with thecytotoxic effects that NK cells expressing certain constructs exhibit.

In accordance with several embodiments disclosed herein, multiplesignaling regions may be used. Additional experiments were conducts toevaluate the expression of a NKG2D-CD16-41BB in expanded NK cells(experiments were conducted using cells from one donor). The expressiondata is shown in FIGS. 12A-12B. FIG. 12A shows raw flow cytometry datathat demonstrate that the addition of the 4-1BB signaling region doesnot significantly impair the expression of the construct by NK cells, ascompared to the NKG2D-CD16 construct. This is also reflected in thesummary histogram of FIG. 12B that shows the relative amount of NKG2Dreceptors on the surface of each of the NK cell groups tested. TheNKG2D-CD16-41BB shows slightly reduced MFI as compared to NKG2D-CD16,but both constructs show elevated expression versus mock.

Cytotoxic effects were evaluated as described above, using both REH andU-2 OS cells as targets. FIGS. 13A-13B depict the resultant data (errorbars represent standard deviations; all experiments were conducted intriplicates, n=3). FIG. 13A shows the cytotoxic effects of theconstructs against REH cells. Similar to the experiment above, theNKG2D-CD16-expressing cells shown with an arrow labeled (b)) did notshow significantly elevated cytotoxic effects as compared to mock NKcells shown with an arrow labeled (a). In contrast, NK cells expressingNKG2D-CD16-41BB (shown with an arrow labeled (c)) showed enhancedcytotoxicity against REH cells. With respect to efficacy against U-2 OScells, both the NKG2D-CD16 and NKG2D-CD16-41BB expressing cells showedenhanced cytotoxicity, with the NKG2D-CD16-41BB expressing cellsexhibiting a more robust cytotoxic effect. This demonstrates that, inaccordance with several embodiments, use of a combination of signalingdomains can result in unexpected enhancements in the efficacy of atransduced NK cell. Thus, as described above, several embodiments employtwo or more transmembrane/signaling domains that work synergisticallytogether to yield enhanced cytotoxicity against target cells.

Example 3—Additional NKG2D Constructs

Additional constructs with varying extracellular domains, transmembranedomains, and intracellular effector domains were generated to evaluatetheir expression and cytotoxicity. The 12 constructs generated forevaluation in this experiment are schematically shown in FIG. 14. Someof these variant chimeric receptors rely on a CD16 transmembrane regionto associate with either CD3ζ or FcRγ. As discussed above, in severalembodiments, the constructs employed rely on endogenous expression ofCD3ζ or FcRγ, however, in several embodiments the plasmid encoding thechimeric receptor (or a separate plasmid) is configured to elevateexpression of CD3ζ and/or FcRγ by the NK cell, thereby enhancing thepotency of the cells. As above, expression levels of the constructs wereevaluated. Mock-transfected NK cells show low levels of NKG2D expressionas evaluated by MFI (FIG. 16A). In contrast, NK cells transduced withthe variant NKG2D constructs described above showed varying levels ofNKG2D expression, with engineered variant constructs 4 and 9 exhibitingsignificantly enhanced expression in NK cells. FIG. 16B depictsrepresentative flow cytometry data for variant NKG2D constructs 1, 4, 8,9 after transduction into the NK cells of two donors. Relative tomock-transduced NK cells, Variant 8- and 9-transduced NK cells showedparticularly strong expression of the chimeric receptor. Variantconstruct expression persisted in the NK cells of two donors 7 daysfollowing transduction, with Variants 8 and 9 showing particularlyelevated levels as evaluated by MFI (FIG. 16C). These data are importantto demonstrate that the constructs can effectively be introduced into NKcells and are expressed. Having established expression of theconstructs, their ability to deliver cytotoxic effects in transduced NKcells was also evaluated. The cytotoxicity of the NKG2D variantconstructs 4, 8, and 9 were evaluated 14 days post-transduction into NKcells at a 1:1 E:T ratio (FIG. 17).

Further variant constructs were generated and are schematically shown inFIG. 15, which show the structure of chimeric receptors comprisingvarious extracellular domains, transmembrane domains, and intracellulareffector domains. Some of these variant chimeric receptors rely on aneffector domain comprising CD3zeta and/or another signaling domain totransduce signaling upon ligand binding, while other variant chimericreceptors comprise a CD3zeta transmembrane domain that recruitsfull-length CD3zeta molecule to the synapse via dimerization. As above,expression levels of the constructs were evaluated. As evaluated by MFI(FIGS. 18A-B), NK cells transduced with engineered constructs exhibitedincreased expression of the chimeric receptor relative to mocktransduced cells. Cytotoxic effects were evaluated as described aboveusing an effector: target ratio of 1:1. As depicted in FIGS. 19A-B, NKcells transduced with engineered constructs (particularly variant 18)have enhanced cytotoxicity relative to the mock control.

As variant 18 exhibited robust expression in NK cells that wasaccompanied by enhanced cytotoxic effects, a series of variant NKG2Dconstructs comprising a CD3zeta transmembrane domain were generated.These variants are termed “NK39” and are schematically shown in FIG. 15.Fourteen days following transfection into donor NK cells (with 4 days ofculturing in low IL-2 conditions), the cytotoxicity of the transduced NKcells were evaluated. FIG. 21 shows the cytotoxic effects of theconstructs against cultured REH cells at 1:1 and 1:2 E:T ratios. All theof the NK cells expressing engineered NK39 constructs showedsignificantly elevated cytotoxic effects as compared to control NK cellsat a 1:1 E:T ratio. When evaluated at a 1:2 E:T ratio, chimericconstructs 16-7, 39-1, 39-2, 39-3, and 39-5 each enhanced the cytotoxiceffects of their respective transduced NK cells relative to the mockcontrol. As exogenous expression of activating receptors can lead to NKcell anergy and cell death, the engineered constructs were transducedinto two donor NK cells and survival was evaluated after 21 days. Asdepicted in FIGS. 23A-B, NK39-5 and NK39-10 transduced cells show bettersurvival than NK16 in two tested donors.

Example 4—Evaluation of NK45 NKG2D Constructs

Additional constructs with varying extracellular domains, hinges,transmembrane domains, and intracellular effector domains according toembodiments disclosed herein are schematically shown in FIG. 22. Theexpression, cytotoxicity, persistence, and cytokine production mediatedby these 7 constructs were evaluated in this Example relative to threeof the NK39 constructs described in Example 3 (NK39-5, NK39-6, NK39-10)as well as a version of NK16 that bicistronically expressesmembrane-bound interleukin 15 (NK26-8). In accordance with severalembodiments disclosed herein, multiple signaling regions may be used.Some of these variant chimeric receptors rely on an effector domaincomprising CD3zeta and/or another signaling domain (e.g., OX40, CD28,and/or 4-1BB costimulatory domains) to transduce signaling upon ligandbinding, while other variant chimeric receptors comprise a CD3zetatransmembrane domain that recruits full-length CD3zeta molecule to thesynapse via dimerization. As disclosed herein, these constructs arefurther configured to co-express membrane-bound IL15.

As above, the ability of NK cells to effectively express theseconstructs was first assessed. NK cells expanded from the PBMC of fourdonors were transduced with the variant constructs (or an empty MSCVcontrol vector containing GFP only) and NKG2D expression was evaluatedby MFI after 3 days. As depicted in FIG. 24, mock-transfected NK cellsshow relatively low levels of NKG2D expression. In contrast, theengineered variant constructs exhibited significantly enhancedexpression, with NK45-4 (NKG2D-OX40-CD3ζ) showing surprisingly robustexpression in all donors. OX40 is expressed in activated NK cells, butits role has not been well-established. A variant chimeric receptor withan effector domain containing a CD28 costimulatory domain (NK45-2;NKG2D-CD28-CD3ζ) also demonstrated robust expression 3 dayspost-transduction.

Having established expression of the variant constructs, their abilityto exert cytotoxic effects was evaluated as above using REH and HL60cells as targets. The potency of NK cells from four donors were examinedagainst REH cells (FIG. 25A) and HL60 cells (FIG. 25B) at 1:1 E:T ratios14 days post-transduction. As depicted in FIGS. 25A-B, the engineeredconstructs exerted an enhanced cytotoxicity against both REH and HL60cells in all four donors as compared to mock NK cells. In addition toits pronounced expression profile, cells expressing NK45-4(NKG2D-OX40-CD3ζ) also exhibited surprisingly elevated cytotoxicityrelative to the mock control and the other constructs tested. NK cellsexpressing NK45-1 and NK45-2 also demonstrated pronounced cytotoxicityin these assays. These data demonstrate that, in accordance with severalembodiments, use of a combination of signaling domains (particularly anOX40 costimulatory domain) can result in unexpected enhancements in theefficacy of a transduced NK cell. FIGS. 28A-B depict the cytotoxicactivity against U2OS cells of the NK cells transduced with several ofthe variant constructs at various E:T ratios (1:2 and 1:4) and assessedover a more extended period of time. Surprisingly, NK cells transducedwith the 45-4 construct appear to maintain cytotoxic activity throughthe time course. Advantageously, these experiments indicate that,according to several embodiments disclosed herein, the NKG2D variantconstructs provide unexpectedly enhanced cytotoxicity over an extendedperiod of time, which, depending on the embodiment, can range from 2-3days, 3-5 days, 5-7, days, 7-8 days, 8-10 days, 10-14 days, 14-21 days,or 21-50 days (and any range in between those listed, includingendpoints). In several embodiments, even longer durations of cytotoxiceffects are achieved.

Further to the cytotoxicity data, the mechanism by which the NK cellsare exerting these effects was examined by evaluating their productionof IFNγ, TNFα, and GM-CSF following stimulation with REH cells. Asdepicted in FIGS. 26A-C, expression of each of the variant constructsyielded enhanced cytokine secretion relative to the production of IFNγ,TNFα, and GM-CSF exhibited by the GFP-expressing control NK cells. Thechimeric receptor NK45-1 consistently mediated high cytokine production,which is surprising because this construct expresses at substantiallylower levels than NK26-8 (from which it differs only with regards to thehinge region). Thus, these data demonstrate the unexpected importance ofthe hinge regions disclosed herein to mediating robust cytokineproduction in response to stimulation. Additionally,NKG2D-OX40-CD3ζ-expressing NK cells also showed an elevated productionof IFNγ, TNFα, and GM-CSF.

As exogenous expression of activating receptors can lead to NK cellanergy and cell death, the engineered constructs were transduced intotwo donor NK cells and the total cell count was evaluated 7, 14, and 21days post-transduction. Surprisingly, the unexpectedly robust expressionof NK45-4 does not come at the cost of reduced NK cell persistence inculture, as the total cell count remained at levels comparable to theGFP-expressing control cells (FIGS. 27A and 27B). Likewise, other NKcells expressing variant constructs at high levels continued toproliferate in the 2 donors for at least 3 weeks post-transduction.Collectively, these data demonstrate that, in accordance with severalembodiments disclosed herein, engineered constructs can successfully beexpressed at high levels in NK cells and mediate cytotoxic effects, andfurther, that this enhanced expression does not come at the detriment ofreduced NK cell proliferation and/or survival.

It is contemplated that various combinations or subcombinations of thespecific features and aspects of the embodiments disclosed above may bemade and still fall within one or more of the inventions. Further, thedisclosure herein of any particular feature, aspect, method, property,characteristic, quality, attribute, element, or the like in connectionwith an embodiment can be used in all other embodiments set forthherein. Accordingly, it should be understood that various features andaspects of the disclosed embodiments can be combined with or substitutedfor one another in order to form varying modes of the disclosedinventions. Thus, it is intended that the scope of the presentinventions herein disclosed should not be limited by the particulardisclosed embodiments described above. Moreover, while the invention issusceptible to various modifications, and alternative forms, specificexamples thereof have been shown in the drawings and are hereindescribed in detail. It should be understood, however, that theinvention is not to be limited to the particular forms or methodsdisclosed, but to the contrary, the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the various embodiments described and the appended claims.Any methods disclosed herein need not be performed in the order recited.The methods disclosed herein include certain actions taken by apractitioner; however, they can also include any third-party instructionof those actions, either expressly or by implication. For example,actions such as “administering a population of expanded NK cells”include “instructing the administration of a population of expanded NKcells.” In addition, where features or aspects of the disclosure aredescribed in terms of Markush groups, those skilled in the art willrecognize that the disclosure is also thereby described in terms of anyindividual member or subgroup of members of the Markush group.

The ranges disclosed herein also encompass any and all overlap,sub-ranges, and combinations thereof. Language such as “up to,” “atleast,” “greater than,” “less than,” “between,” and the like includesthe number recited. Numbers preceded by a term such as “about” or“approximately” include the recited numbers. For example, “about 90%”includes “90%.” In some embodiments, at least 95% homologous includes96%, 97%, 98%, 99%, and 100% homologous to the reference sequence. Inaddition, when a sequence is disclosed as “comprising” a nucleotide oramino acid sequence, such a reference shall also include, unlessotherwise indicated, that the sequence “comprises”, “consists of” or“consists essentially of” the recited sequence.

1.-132. (canceled)
 133. A polynucleotide encoding a chimeric receptorcomprising: (a) an extracellular receptor domain, wherein saidextracellular receptor domain comprises a peptide that binds nativeligands of Natural Killer Group 2 member D (NKG2D), wherein the peptidethat binds native ligands of NKG2D is a fragment of NKG2D, wherein thefragment of NKG2D is encoded by a polynucleotide comprising: (i) afragment of SEQ ID NO: 1, (ii) SEQ ID NO. 2, or (iii) SEQ ID NO. 3; andan effector domain comprising a transmembrane region and anintracellular signaling domain, wherein the transmembrane regioncomprises a CD8a hinge domain and a CD8a transmembrane domain, andwherein the intracellular signaling domain comprises an OX-40 domain andCD3zeta.
 134. The polynucleotide of claim 133, wherein thepolynucleotide further encodes, in a bicistronic configuration, amembrane-bound interleukin 15 (mbIL15).
 135. The polynucleotide of claim134, wherein the fragment of NKG2D is encoded by a polynucleotidecomprising SEQ ID NO. 2 and wherein the mbIL15 is encoded by SEQ ID NO.16.
 136. The polynucleotide of claim 134, wherein the chimeric receptoris encoded by the nucleic acid sequence of SEQ ID NO: 90 and wherein thembIL15 is encoded by SEQ ID NO.
 16. 137. The polynucleotide of claim136, wherein the CD3zeta is encoded by a polynucleotide having at least98% homology to the polynucleotide of SEQ ID NO.
 13. 138. Thepolynucleotide of claim 137, wherein the encoded chimeric receptorcomprises the amino acid sequence of SEQ ID NO: 91 and wherein theencoded mbIL15 comprises SEQ ID NO:
 17. 139. The polynucleotide of claim134, wherein the fragment of NKG2D is encoded by a polynucleotidecomprising SEQ ID NO. 3 and wherein the mbIL15 is encoded by SEQ ID NO.16.
 140. A polynucleotide encoding a chimeric receptor, comprising: (a)an extracellular receptor domain, wherein said extracellular receptordomain comprises a peptide that binds native ligands of Natural KillerGroup 2 member D (NKG2D), wherein the peptide that binds native ligandsof NKG2D is a fragment of NKG2D, wherein the fragment of NKG2D isencoded by a polynucleotide comprising SEQ ID NO. 2, and (b) an effectordomain comprising a transmembrane region and an intracellular signalingdomain, wherein the transmembrane region comprises a CD8a transmembranedomain, wherein the intracellular signaling domain comprises an OX40domain and a CD3zeta.
 141. The polynucleotide of claim 140, wherein thepolynucleotide further encodes a membrane-bound interleukin 15 (mbIL15).142. The polynucleotide of claim 140, wherein the mbIL15 is encoded by apolynucleotide comprising SEQ ID NO. 16 and wherein the CD3zeta isencoded by a polynucleotide having at least 98% homology to thepolynucleotide of SEQ ID NO.
 13. 143. The polynucleotide of claim 140,wherein the chimeric receptor is encoded by the nucleic acid sequence ofSEQ ID NO:
 90. 144. The polynucleotide of claim 140, wherein thechimeric receptor comprises the amino acid sequence of SEQ ID NO: 91.145. A polynucleotide encoding a chimeric receptor, comprising: (a) anextracellular receptor domain, wherein said extracellular receptordomain comprises a peptide that binds native ligands of Natural KillerGroup 2 member D (NKG2D), wherein the peptide that binds native ligandsof NKG2D is a fragment of NKG2D, wherein the fragment of NKG2D isencoded by a polynucleotide comprising SEQ ID NO. 2, and (b) an effectordomain comprising a transmembrane region and an intracellular signalingdomain, wherein the intracellular signaling domain comprises CD3zeta,and wherein the CD3zeta is encoded by a polynucleotide having at least98% homology to the polynucleotide of SEQ ID NO. 13, and wherein thepolynucleotide further encodes a membrane-bound interleukin 15 (mbIL15).146. The polynucleotide of claim 145, wherein the transmembrane regionof the effector domain comprises a CD8a transmembrane domain, whereinthe mbIL15 is encoded by a polynucleotide comprising SEQ ID NO. 16, andwherein the effector domain further comprises an OX-40 domain.
 147. Thepolynucleotide of claim 146, wherein mbIL15 comprises an amino acidsequence of SEQ ID NO:
 17. 148. The polynucleotide of claim 146, whereinthe chimeric receptor comprises the fragment of NKG2D coupled to a CD8ahinge, the CD8a transmembrane domain, the OX-40 domain, and the CD3zeta.149. The polynucleotide of claim 148, wherein the CD8a hinge region isencoded by a polynucleotide comprising SEQ ID NO:
 5. 150. Thepolynucleotide of claim 146, wherein the chimeric receptor is encoded bythe nucleic acid sequence of SEQ ID NO: 90 and is coupled topolynucleotide encoding the mbIL15 of SEQ ID NO.
 16. 151. Thepolynucleotide of claim 150, wherein the chimeric receptor comprises theamino acid sequence of SEQ ID NO: 91 coupled to the mbIL15 comprisingthe amino acid sequence of SEQ ID NO.
 17. 152. The polynucleotide ofclaim 146, wherein the mbIL15 is bicistronically expressed on the samepolynucleotide as the chimeric receptor.